1.

INTRODUCTION
Multiplex PCR is a techniques by which we can identify particular gene of any bacteria, any
cells or any type of organism by targeted their gene with with the specific primer, in this
technique we can use multiple of DNA and can amplify each DNA with multiple of primers at
one time to rapid identify multiple of organism from given population of samples. Traditionally,
detection and enumeration of bacterial pathogen have been largely based on the use of selective
culture and standard biochemical method(Kong et al,).But these methods suffer from numbers
of drawbacks- Firstly, Pathogenic bacteria which normally occurs in low numbers tend to incur
large number of error in sampling and enumeration(Fleisher JM). Secondly, culture based
method are time consuming, tedious, invariably monospecific (i.e. detecting low output).
Thirdly,many pathogenic organism in the environment although viable, are either difficult to
culture or non-culturable (Fleisher JM), but can still cause illness (Rahman et al,1996). Due to
this difficulties, examination of water samples for pathogens like vibrio cholerae, shigella
dysenteriae, aeromonas spp. and campylobacter spp.,etc. is normally not performed during
routine microbiological assesment of water quality (Kong et al,). The aim of this study is to
screen different water bodies (tap water,pond water, river water and fish water) for the presence
of vibrio species and determine their pathogenicity. The knowledge will help to control vibrioassociated gastroentritis in india as the awareness of the danger associated with the consumption
of tap water, raw and undercooked seafood will be created for the masses. Vibrio are gramnegative, straight,curved rod,non spore forming,motile,usually contain single polar flagellum
and halophilic,few species are non-halophilic those who are halophic they require salt
medium(Nacl) for their growth. Mostly are sensitive to acidic pH,and few are alkaline pH
tolerant. According to centre of disease control 8,000 infection and 60 death happens in each
year by vibrio infection. Till now date vibrio have 72 species all around the world, out of 72
twelve account for the majority of vibrio infection in humans.vibrio are water surface organism
occurs in fresh water as well as salt water(river,pond,fish and industrial effluent etc..) they are
pathogen of marine living organism,this is the reason that doctor suggested do not to eat
uncooked food (fish,lobster,crack) becouse comsuming of uncooked food is the indirect
pathway of vibrio to enter inside the human body.

1 | Page

V.cholerae,V.parahaemolyticus,V.vulnificus,V.alginolyticus,V.mimicus,V.fluvialis,V.adaptatus,V.c
ampbellii,V.diabolicus,V.fischeri,V.furnissii,V.tapetis V.hollisae, V.damsela and many more
species like this are human pathogen. Most of this species secrete enterotoxins in foods,water
and gastrointestinal tract. Identification of vibrio species is very important for us becouse this
are the main cause of human infection. If we could identify that water bodies which are
contaminated by vibrio so that we can make them free from pathogens and prevent to cause any
water born disease again.
A multiplex polymerase chain reaction (PCR) method,specifically designed for application in
routine diagnostic laboratories, was developed for identifying 5 human pathogenic Vibrio
species:

V.cholerae,V.parahaemolyticus,V.vulnificus,V.alginolyticus,V.mimicus.

This

assay

directed toward the dnaJ gene ( a housekeeping gene that encodes heat shock protein 40,for the
identification of vibrio species) was tested on the total of 38 strains representing 23 vibrio
species. Specific PCR fragments were formed in isolates which belong to the 5 target species
and were absent from all strains other than this 5 species, indicating high specificity of multiplex
PCR. This technique represented a rapid detection of the 5 major pathogenic vibrio species.
Most of the effort for vibrio identification were done with marine water, Now the present study
based on rapid identification of vibrio species form marine water as well as fresh water.
Colour of vibrio species colonies on TCBS media
Vibrio alginolyticus

Yellow

Vibrio vulnificus

Green 85% or Yellow 15%

Vibrio cholera

Yellow

Vibrio mimicus

Green

Vibrio parahaemolyticus

Blue to Green centered colony

2 | Page

Objectives
1. Isolation and characterization of vibrio species from tap water, pond water, fish water
and river.
2. Isolation of DNA from the vibrio colonies obtained on the TSA media.
3. Qualitative determination of the isolated DNA using Agarose Gel Electrophoresis
4. Quantification of DNA using UV Double beam Spectrophotometer.
5. To confirm the pathogenic species by specific primer design from its toxin genes by
PCR.
6. Identification of human pathogenic vibrio species by multiplex PCR.

3 | Page

2. REVIEW OF LITERATURE
Ozaki et al.(2014)observed that the regional differences in human papillomavirus (HPV)
genotypes and the presence of mixed HPV infections may affect adversely the efficacy of the
HPV vaccine. Therefore, a simple and high-throughput HPV genotyping system is required.
Recently, a novel HPV genotyping kit (the Mebgen TMHPV kit) was developed. This kit uses
multiplex PCR and Luminex format. In the present study, the analytical performance of the
kitwas examined using HPV x MAPTM technology to detect 13 types of high-risk HPVs and an
internal control in a 96-well plasmid DNA. All 13 types of HPVs were detected with a minimum
detection sensitivity of 250 copies/test, and highly specific signals were observed. HPV 16
plasmid was detected in samples containing mixtures with other HPV-type plasmids in ratios
ranging from 1:1 to 1:1000. No cross reactivity was observed with DNA from 27 types of other
infectious microbes. A clinical evaluation was carried out using cervical samples from 356
patients with persistent abnormal smears diagnosed atmass public health screenings for cervical
cancer. The samples were preserved in Tacas TM medium until analysis. HPV was detected in
162 (45.5%) samples including 110 (67.9%) with single infections and 52(32.1%) with multiple
infections. The type distribution of the 13 high-risk HPV was as follows: 28.4%HPV 16, 11.7%
HPV 18, 6.8% HPV 31, 3.1% HPV 33, 3.7% HPV 35, 9.3% HPV 39, 1.9% HPV 45, 8.6% HPV
51,37.0% HPV 52, 9.3% HPV 56, 16.7% HPV 58, 3.7% HPV 59, and 1.9% HPV 68. To
evaluate sample stability over time, changes in the detection of HPV DNA derived from HeLa
and SiHa cells were measured in 3 types of liquid-based cytology media. HPV DNA was
detected in Tacas and Thinprep TM samples after storage at 4C or 30C for 4 weeks and within
1 week of collection in Surepath TM samples. These results suggest that this newly developed
HPV genotyping kit is suitable for use in both clinical applications and large-scale
epidemiological studies.
Gosiewskil

et al.(2014) described about the application of the PCR method for the

simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi

and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may
cause sepsis. Material for the study was sterile blood inoculated with four species of
microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus
4 | Page

fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed
method is based on nested-multiplex real-time PCR .Analysis of the obtained data shows that
sensitivity of nested-multiplex real-time PCR remained at the level of 101 CFU/ml for each of
the four studied species of microorganisms and the percentage of positive results of the
examined blood samples from the patients was 70% and 19% for the microbiological culture
method. The designed primers correctly typed the studied species as belonging to the groups of
Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi. Results
obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole
blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the
main groups of microorganisms within a few hours.
Kamau et al.(2014) reported that microscopy and antigen detecting rapid diagnostic tests are
the diagnostic tests of choice in management of clinical malaria. However, due to their
limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident
as more studies are now utilizing molecular methods in detection of malaria. Some of the
challenges that continue to limit the widespread utilization of qPCR include lack of assay
standardization, assay variability, risk of contamination, and the need for cold-chain.
Lyophilization of molecular assays can overcome some of these limitations and potentially
enable widespread qPCR utilization. A recently published multiplex malaria qPCR assay was
lyophilized by freezing drying into Sample-Ready format (MMSR). MMSR assay contained
all the required reagents for qPCR including primers and probes, requiring only the addition of
water and sample to perform qPCR. The performance of the MMSR assay was compared to the
non-freeze dried, wet assay. Stability studies were done by maintaining the MMSR assays at
four different ambient temperatures of 4C, room temperature (RT), 37C and 42C over a
period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax
DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two
different concentrations. The CT values and the standard deviations (SD) were used in the
analysis of the assay performance.The limit of detection for the MMSR assay was 0.244
parasites/L for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to
wet assay which was 0.39 and 3.13 parasites/L for PLU and FAL assay targets, respectively.
The MMSR assay performed with high efficiencies similar to those of the wet assay and was
5 | Page

stable at 37C for 42 days, with estimated shelf-life of 5 months. When used to analyse field
clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the
wet assay.The MMSR assay has the same robust performance characteristics as the wet
assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option
in choosing assay for malaria diagnostics depending on the application, needs and budget.
Gautier et al.(2014) observed that zymoseptoria tritici is a hemibiotrophic ascomycete fungus
causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are
known to be highly diverse and poorly differentiated from each other. However, a genotyping
tool is needed to address further questions in large collections of isolates, regarding regional
population structure, adaptation to anthropogenic selective pressures, and dynamics of the
recently discovered accessory chromosomes. Procedure is limited by costly and time-consuming
simplex PCR genotyping. Recent development of genomic approaches and of larger sets of
SSRs enabled the optimization of microsatellite multiplexing.A reliable protocol to amplify 24
SSRs organized in three multiplex panels, and covering all Z. tritici chromosomes. We also
propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable
manner across studies and laboratories. All together, tools enabled us to characterize local and
worldwide populations and to calculate diversity indexes consistent with results reported in the
literature.Easy-to-use, accurate, repeatable, economical, and faster technical strategy can
provide useful genetic information for evolutionary inferences concerning Z. tritici populations.
Moreover, it will facilitate the comparison of studies from different scientific groups.
Arreola et al.(2014) reported that the sophisticated methodologies are available, the use of
endpoint polymerase chain reaction(PCR) to detect 16S rDNA genes remains a good approach
for estimating the incidence and prevalence of specific infections and for monitoring infections.
Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the
development of a sensitive and affordable method for identifying pathogens in clinical samples
is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (mPCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause
genital infections, and the PCR method was developed. The Genosensor Probe Designer (GPD)
(version 1.0a) software was initially used to design highly specific and efficient primers for in6 | Page

house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers
for each locus in turn were added individually in subsequent amplifications until m-PCR was
achieved. Amplicons of the expected size were obtained from each of the four bacterial gene
fragments. Finally, the analytical specificity and limits of detection were tested. Because they
did not amplify any product from non-STI tested species, the primers were specific. The
detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis
and Ureaplasma urealyticum primer sets were 5.12 105, 3.9103 , 61.19 106 and 6.37 105
copies of a DNA template,respectively.The methodology designed and standardised here could
be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis,
Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. Method is at least
as efficient as other previously described methods; however, this method is more affordable for
low-income countries.
Wu et al.(2014) reported that the novel method to simultaneously detect expression of four
genes, ribonucleotide reductase subunit M1(RRM1), X-ray repair cross-complementing gene 1
(XRCC1), thymidylate synthase (TS) and class III -tubulin (TUBB3), and to assess their
application in the clinic for prediction of response of non-small cell lung cancer (NSCLC) to
chemoradiotherapy. Designed four gene molecular beacon (MB) probes for multiplex
quantitative real-time polymerase chain reactions to examine RRM1, XRCC1, TUBB3 and TS
mRNA expression in paraffin-embedded specimens from 50 patients with advanced or
metastatic carcinomas. Twenty one NSCLC patients receiving cisplatin-based first-line
treatment were analyzed. Molecular beacon probes could specially bind to their target genes in
homogeneous solutions. Patients with low RRM1 and XRCC1 mRNA levels were found to have
apparently higher response rates to chemoradiotherapy compared with those with high levels of
RRM1 and XRCC1 expression (p<0.05). The TS gene expression level was not significantly
associated with chemotherapy response (p>0.05).A method of simultaneously detecting four
molecular

markers

was

successfully

established

and

applied

for

evaluation

of

chemoradiotherapy response. It may be a useful tool in personalized cancer therapy.

Jeong et al.(2014) observed that the wide range of inter-individual variations in platelet
responses to clopidogrel. The variations in response to clopidogrel can be driven by genetic
polymorphisms involved in the pathway of absorption, distribution, metabolism, excretion, and
7 | Page

the target receptor P2Y12. A set of genetic variants known for causing variations in clopidogrel
responses was selected, which included CYP2C19*2, *3, *17, CYP2B6*4, *6, *9, CYP3A4*18,
CYP3A5*3, MDR1 2677G > T/A, 3435C > T, and P2Y12 H2 (742T > C). The simultaneous
detection of these 10 variants was developed by using a multiplex PCR and single-base
extension (MSSE) methodology. The newly developed genotyping test was confirmed by direct
DNA sequencing in the representative positive control samples and validated in an extended set
of 100 healthy Korean subjects. Genotyping results from the developed MSSE exhibited a
perfect concordance with the direct DNA sequencing data and all of variants tested in 100
healthy Korean subjects were in agreement with Hardy-Weinberg equilibrium (p > 0.05). The
present molecular diagnostic studies provide an accurate, convenient, and fast genotyping
method for the detection of multiple variants helpful for researchers, as well as clinicians, to use
genetic information toward more personalized medicine of clopidogrel and other antiplatelet
drugs in the future.
Qiu et al,(2014) study that the Detection of hepatitis viral infections has traditionally relied on
the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex
real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has
proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus
(HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a
main public health problem. A one-step multiplex reverse transcriptase quantitative polymerase
chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV
and HEV. This novel detection system proved specific to the target viruses, to be highly
sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and
feasible identification of HAV and HEV.
Corach et al.(2014) reported that the microdeletions in the AZF region of the Y chromosome
are among the most frequent genetic causes of male infertility, although the specific role of the
genes located in this region is not fully understood. AZFa and AZFb deletions impair
spermatogenesis since no spermatozoa are found in the testis. Deletions of the AZFc region,
despite being the most frequent in azoospermic patients, do not correlate with spermatogenic
failure. Therefore, the aim of work was to develop a screening method to ascertain the presence
8 | Page

of the main spermatogenesis candidate genes located in the AZFc region in the light of the
identification of those responsible for spermatogenic failure. DAZ, CDY, BPY2, PRY,
GOLGA2LY and CSGP4LY genes were selected on the basis of their location in the AZFc
region, testis-only expression, and confirmed or predicted protein codification. AMEL and SRY
were used as amplification controls. The identification of Real Time PCR products was
performed by High Resolution Melting analysis with SYTO 9 as intercalating dye. The herein
described method allows a rapid, simple, low-cost, high-throughput screening for deletions of
the main AZFc genes in patients with spermatogenic failure. This provides a strategy that would
accelerate the identification of spermatogenesis candidate genes in larger populations of patients
with non-obstructive idiopathic azoospermia.
Hofler

et al.(2014) study that the

federation of European Laboratory Animal Science

Association (FELASA) recommends screening of laboratory rodents and biological materials

for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date
for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform.
However, an infection even if silent alters experimental results through changing the animals
physiology and increases inter-individual variability. As a consequence higher numbers of
animals and experiments are needed for valid and significant results developed a novel highthroughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous
identification of 24 DNA viruses infecting mice and rats and detected all 24 DNA viruses with
high specificity and reproducibility. Detection limits for the different DNA viruses varied
between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated
from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The
biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes
coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have
the potential to replace conventional testing and may simplify and improve routine detection of
DNA viruses infecting rodents.
Velasco et al.(2014) study was to compare a real-time PCR assay, with a conventional
culture/PCR method, to detect S. aureus,mecA and Panton-Valentine Leukocidin (PVL) genes in
animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples
9 | Page

were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex
real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with
methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment
samples were assessed. The conventional culture/PCR method included the two-step selective
enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The
conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of
real-time PCR on samples following primary and secondary enrichment detected S. aureus in
111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was
0.680.88 (from substantial to almost perfect agreement) and 0.290.77 (from fair to substantial
agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA
gene, the kappa statistic was 00.49 (from no agreement beyond that expected by chance to
moderate agreement) for primary and secondary enrichment samples. Two pork samples were
mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples
that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not
detected in any sample by the conventional culture/PCR method or the real-time PCR assay.
Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and
multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR
assay may be recommended as a rapid method for detection of S. aureus and the mecA gene,
with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture
method.
Gimenes et al.(2014) observed that the sexually transmitted diseases (STDs) may impair sperm
parameters and functions thereby promoting male infertility. To date limited molecular studies
were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed
at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of
the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae,
Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) 21 and 22, and
Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in
screening programs for semen pathogens. In addition, we aimed: to detect human
Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to
determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility
10 | P a g e

that these infections affect semen parameters and thus fertility. The overall validation parameters
of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity
(99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%).
The prevalence of STDs was very high (55.3%). Furthermore, associations were observed
between STDs and changes in semen parameters, highlighting the importance of STD detection
in semen. Thus, this M-PCR assay has great potential for application in semen screening
programs for pathogens in infertility and STD clinics and in sperm banks.
Boudreau

et al.(2014) observed that the among NK cell receptor-ligand partnerships,

KIR3DL1 and HLA-Bw4 demonstrate the greatest diversity; permutations of their allelic
combinations titrate NK reactivity. Balancing selection has maintained distinct subtypes of
KIR3DL1 alleles in global populations, implying that each may provide unique fitness
advantages and variably influence disease processes. Though approaches exist for determining
HLA-B allotypes, practical methods for identifying KIR3DL1 alleles are lacking. We have
developed a PCR-based approach that identifies functional subtypes of KIR3DL1 alleles; it is
suitable for research and may have clinical application. Six allele subsets were identified based
on expression characteristics of the eleven most common KIR3DL1 alleles represented in
reported populations. The remaining 62 low-frequency alleles were distributed into these groups
based on sequence homology to coding regions. Subtype-specific SNPs were found in exons 3,
4, and 7, and used as priming sites for five multiplex PCR reactions. Genomic DNA derived
from 175 unrelated donors and 52 related individuals from 6 families demonstrated .99.5%
concordance between sequence-based typing and our novel approach. Finally, PCR-based typing
accurately predicted NK phenotypes obtained by flow cytometry after staining with DX9 and
Z27 monoclonal antibodies. This novel approach facilitates high-throughput analysis of
KIR3DL1 allotypes to enable a broader understanding of KIR3DL1 and HLA-Bw4 interaction
in health and disease.
Chung et al.(2014) was investigate whether tissue samples processed by the rapid urease test
(RUT) kit are suitable for dualpriming oligonucleotide-based multiplex polymerase chain
reaction (DPO-PCR) to detect Helicobacter pylori (H. pylori ). A total of 54 patients with
specific gastrointestinal symptom were enrolled in this study. During endoscopy, gastric biopsy
11 | P a g e

specimens were taken for histology, RUT, and DPO-PCR. DPO-PCR was performed on gastric
biopsy samples and tissue samples that were analyzed by RUT at 2 separate institutes. In
detecting H. pylori , the concordance rate of the DPOPCR tests between the tissue samples that
had been submitted to RUT and the gastric biopsy samples was investigated. H. pylori cooccurred with 76.0% (19/25) of gastric ulcers, 64.3% (9/14) of duodenal ulcers, and 33.3%
(4/12) of gastritis cases. H. pylori infection was found in 100% (3/3) of the patients with both
gastric and duodenal ulcers. Overall, H. pylori was detected in 35 of 54 (64.8%) patients. The
diagnostic sensitivities of histology, RUT, and DPO-PCR were 85.7% (30/35), 74.3% (26/35),
and 97.1% (34/35), respectively (P = 0.02). The positive predictive value (PPV) of DPO-PCR
was 94.4%, whereas the negative predictive value (NPV) was 94.7%. In the rapid urease test
(CLOtest)-negative cases, the frequency of positive DPO-PCR and histologic results was 20.0%
(7/35). The concordance rate of the DPO-PCR tests between the tissue samples from the RUT
kit and the gastric biopsy samples was 94.4% (51/54). The rate of DPOPCR and silver stain
positivity in the RUT-negative cases was 20.0% (7/35).In diagnosing H. pylori infection, DPOPCR can be performed on tissue samples that have been processed by the RUT kit. Particularly,
in patients with RUT-negative results, DPO-PCR on these tissue samples could be helpful in
detecting of H. pylori infection.
Xu et al.(2014) reported that the identification of human body fluids or tissues through mRNAbased profiling is very useful for forensic investigations.Previous studies have shown mRNA
biomarkers are effective to identify the origin of biological samples.Selected 16 tissue specific
biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues
identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and
Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4
(TGM4) for semen, mucin 4 (MUC4) and human beta defensin1(HBD1) for vaginal secretion,
matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin
4(KRT4)for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for
saliva, statherin (STATH) for nasal secretion,dermcidin (DCD) for sweat and uromodulin
(UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used
in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay,
XCYR1, whichincludes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4,
12 | P a g e

DCD, UMOD, MMP7, TGM4, and STATH) and housekeeping genes [i.e., glyceraldehyde-3phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed.Assay was further validated
with real casework samples and mock samples (with both single source and mixture) and it was
approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory
blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and
urine) in forensic casework samples.

13 | P a g e

3. MATERIAL AND METHODS

3.1 Materials
3.1.1 Reagent Used
3.1.1.1 For Media Preparation
3.1.1.1.1 TCBS Agar (Thiosulfate Citrate Bile Salt Sucrose Agar)-100ml
TCBS agar

8.9 gm

Distilled water

100 ml

3.1.1.1.2 TSA media (Trypticase Soy Agar)-100ml

TSA

3 gm

Agar

1.50 gm

Distilled water

100 ml

3.1.1.1.3 TSB (Trypticase Soy Broth)-100ml

TSA

3 gm

Distilled water

100 ml

3.1.1.2 Reagent for DNA Isolation

TE Buffer solution (pH 8.0)

50X (diluted to 1X)

SDS solution

10%

Phenol:chloroform:

25:24:1

isoamyl Alcohol(PCI)Solution
Propanol
3.1.1.3 Reagent for Agarose Gel Electrophoresis (0.8% Gel)
Agarose

0.8 gm

TAE buffer

50X (diluted to 1X)

Ethidium bromide

3.0 l

14 | P a g e

DNA loading dye

5.0 l

Distilled water

100ml

3.1.1.4 Reagent for Agarose Gel Electrophoresis (1.2% Gel)

Agarose

1.2 gm

TAE buffer

50X (diluted to 1X)

Ethidium bromide

3.0 l

DNA loading dye

5.0 l

Distilled water

100ml

3.1.1.5 DNA Loading Dye

Bromophenol Blue

0.25%

Xylene Cyanol

0.25%

Glycerol

30%

After mixing all this with distilled water dye is stored at 4C.
3.1.1.6 Spectrophotometric Quantification of Genomic DNA
DNA sample

10l

Distilled water

990l

3.1.1.7 Polymerase Chain Reaction (PCR)

Table 1: PCR Components.
Chemical
PCR buffer
dNTP
Primer
Universal forward primer
Reverse
primer
vibrio
alginolyticus
Reverse
primer

Stock
10x
2.5mM
100ppm

Working
2l(1x)
2.5l(0.2mM/L)
1l(8ppm)
1l(8ppm)

vibrio

1l(8ppm)

vunificus
Reverse primer vibrio cholera
Reverse primer
vibrio

1l(8ppm)
1l(8ppm)

mimicus
15 | P a g e

Reverse

primer

vibrio

1l(8ppm)

parahaemolyticus
Taq DNA polymerase
Distilled water
DNA sample
Total

1l
7.5l
5l
20l

3.1.2 Glassware Used

Conical Flask

Petri plate

Reagent Bottle

Beaker

Glass Rod

L-shaped rod

3.1.3 Material Required

Butter paper

Spatula

Conical flask

Cotton

Aluminium foil

Petridish

Micropipette

Microtip

Spreader

Clean film

Inoculum loop

Spirit lamp

Spirit

Test tube

Measuring cylinder

Test tube stand

Microcentrifuge tube

Electrophoresis unit

PCR tubes

Vortex

3.1.4 Required Instrument

16 | P a g e

DNA sample

Electophoresis unit

Centrifuge

Laminar flow

Refrigerator

17 | P a g e

Microwave

Weighing machine

Incubator

Spectrophotometer

Waterbath.

Autoclave

PCR

Vortex

Gel doc

3.1.5 Collected Samples- Water samples (Total - 26 Samples)

3.1.5.1 Tap water samples
1.
2.
3.
4.
5.
6.
7.
8.
9.

Ajamgarh tap water(Gorakhpur).

Jankipuram tap water(Lucknow).
Khurram Nagar tap water(Lucknow).
Gudamba Thana tap water(Lucknow).
CytoGene tap water(Lucknow).
Bakshi Ka Talab tap water(Lucknow).
Fajulaganj tap water(Lucknow).
BBAU university tap water(Lucknow).
Adil Nagar tap water(Lucknow).
10Munshi Pulia tap water(Lucknow).

3.1.5.2 Pond Water

1.
2.
3.
4.
5.
6.

Dhatingra pond water(Lucknow)

Jankipuram pond water(Lucknow).
Bsawan Purwa pond water(Lucknow)
Raitha pond 1st water(Lucknow).
Raitha pond 2nd water(Lucknow).
Bakshi Ka Talab pond water(Lucknow).

3.1.5.3 River Water

1.
2.
3.
4.
5.

Gomti river(Lucknow).
Yamuna river(Delhi).
Hindun river(Delhi).
Ganga river(Haridwar).
Ghagra river(Barabanki).

3.1.5.4 Fish Water

1.
2.
3.
4.
5.

Dhatingra fish water(Lucknow).

Jankipuram fish water(Lucknow).
Munshi Pulia fish water(Lucknow).
Khurram Nagar fish water(Lucknow)
Tadikhana fish water(Lucknow).

18 | P a g e

3.1.6 Primer Sequence for Multiple PCR of Five Pathogenic Species of Vibrio
Table 2: List of Primers used for amplification.
Amplicon

Target species

Primer

Sequence(5-3)

Universal primer
V. alginolyticus
V. vunificus
V. cholera
V. mimicus

Universal F.W primer

Reverse primer
Reverse primer
Reverse primer
Reverse primer

CAGGTTTGYTGCACGGCGAAGA
GATCGAAGTRCCRACACTMGGA
GTACGAAATTCTGACCGATCAA
AGCAGCTTATGACCAATACGCC
YCTTGAAGAAGCGGTTCGTGCA

size
144
412
375
177

V. parahaemolyticus

Reverse primer

TGCGAAGAAGGCTCATCAGAG

96

19 | P a g e

3.2 METHODOLOGY
3.2.1 Collected Samples for Experiment
Total 26 sample were collected for the purpose to done this experiment and each sample
collected from different areas. Out of 26 mostly are collected from Lucknow region and
some sample collected from different areas due to lack of river inLlucknow 2 sample of
river collected from Delhi,1 sample collected from Barabanki 1 from Haridwar and 1
sample of tap water collected from Ajamgarh.
3.2.2 Sample Processing
After collection of the 25 sample the following sample processing was done are as
following methods:
3.2.2.1 Autoclaving of Media
An autoclave is a device used to strilize equipment and supplies by subjecting them to
high pressure saturated steam at 121 C for around 1520 minutes depending on the size
of the load and the contents.
Procedure
Took a labelled washed conical flask.
Weighed the media on weighing machine and then added in conical flask with distilled
water to make total volume and stopped with cotton plug.
Packed petridish with paper and labelled them.
Transferred labeled media (Date, type of media, name of person) to inside the autoclave
chamber.
Closed the autoclave chamber.
Pressure reached at 15 lbs wait 15 minutes then stop the chamber.
Take out the sterilized media from autoclave for proceding next steps.
3.2.2.2 Pouring of Media In Petridish
Kept a media inside laminar flow then wash hand with spirit.
Labelled each plate( name,date,Type of media,name of sample).
Pour 25ml autoclved media in each petriplate and leave it to solidify.
3.2.2.3 Spreading of Water Sample on TCBS Media
20 | P a g e

 Took 200l of water sample and put it on TCBS media.

Spreader sterilized by dipping in alcohol and then by exposed with flame and then
allowed to cool.
Spreaded all the inoculum evenly over the surface of TCBS media.
Wrapped plates with clean film then incubated at 28C for 2 days and plates were
examined for bacterial growth.
After incubation,petriplates were taken out from the incubator and bactrial colonies are
visible over the surface of media.
3.2.2.4 Streaking of Bacterial Colony
Streaking is a technique used to isolate a pure strain from a single species of
microorganism, often bacteria. Samples can then be taken from the resulting colonies
and a microbiological culture can be grown on a new plate so that the organism can be
identified, studied, or tested.
Procedure

Red hot inoculum loop and allowed the loop to cooled.

Picked colony from TCBS media.
Streak the inoculm over the surface of TSA media in a zig zag manner.
Labelled each plate with name,date,media,colony colour and sample name.
Wrapped with clean film.
Incubated at 28C for 1 day.

3.2.2.5 Inoculation of Single Colony of Bacteria in Ts Broth

Broth is liquid neutrient medium which has all the component which is required for
bacterial growth,development and division.broth contained casein and soyabean provide
amino acids and nitrogenous source,glucose for energy source,nacl for maintaing
osmotic equilibrium, dipotassium phosphate acts as buffer to maintain pH.
Procedure
Took autoclaved test tubes with labelled name,date,broth type and sample name.
Pour 10 ml autoclaved TS broth in each test tube with the help of sterilized measuring
cylinder.
Inoculate single bacterial colony from TSA media to the TS broth.
21 | P a g e

 Closed opening of test tube with cotton plugs and incubated at 28C for 1 day inside the
incubator.
3.2.2.6 Bacterial DNA Isolation by Phenol Chloroform Method.
The broth which was inoculated with bacteria and incubated for 1 day become the
sample for DNA isolation.
Procedure
From the liquid culture 1.5 ml of culture was taken in microcentrifuge tube and
centrifuge it at 8000 rpm for 5 mins and pellet the bacterial cells.
Resuspended the pellet in 900l of the TE buffer.
Added 1/10 volume of 10% SDS solution(900l solution then add 100l of solution into
it).
Waterbathed the tubes at 50-60C and incubated it for approximately 2 hours.
After 2 hours the solution become thicker then it wass in the starting,now mix 600l of
Phenol:chloroform:isoamyl alcohol(25:24:1)to it and shake gently by repeatedly
inverting the tube.
White precipitate appears when mix this mixture is added to the tube and shaking
solution become creamy orange(white due to protein precipitation and orange due to
phenol).
Kept for 5 min and then centrifuge at 10000 rpm for 10 min.
Upon centrifugation 3 layers appear in tube.
Upper aqueous layer in transparent like water which contain DNA below this is a thin
layer of white colour which is protein precipitate and the lower dark orange layer is
phenol chloroform mixture.
Collected upper transparent aqueous layer in fresh new eppendorf tube and discard the
lower layer.
Kept the eppendorf tube in refrigerator to cool it for 10-15 min.
Added double volume of chilled propanol to the aqueous DNA solution drop by drop
from its wall.after adding DNA precipitate out from the solution and keep it again on 0C
for 15 min,so that DNA is precipitated easily.
After precipitation centrifuge the tube at 10000rpm for 10 min and discard the
supernatant,before discarding mark the DNA pellet with marker on wall of tube.
Dried the DNA pellet in air or by keeping the tube in inverted position on filter paper.
Dissolved the dry DNA pellet in 50l TE buffer.
Checked the quality of DNA isolated by agarose gel electrophoresis.
22 | P a g e

3.2.2.7 Qualitative determination of Bacterial Genomic DNA By Agarose Gel

Electrophoresis (0.8%).
Dissolved agarose in 0.240 gm in 30 ml TAE buffer in a conical flask.
Placed the conical flask in an oven and heat until the solution becomes transparent.
Used hand gloves to remove the conical flask out from oven and wait until it becomes
pal bearable.
Wiped the gel casting tray and comb with ethanol.seal the open Side using cello tape and
set up the apparatus.
Poured the agarose solution in the casting tray.leave to solidify for 15-20 min.
After settling of the gel,mix 3l of gel loading dye with 5l of DNA sample with the

help of a micropipette.
Carefully remove the comb from the solidified gel so that the wells dont break.
Fill the tank with TAE buffer.
Placed the agarose gel in the electrophoresis tank.
The well will be placed toward the cathode.
Loaded the sample in the gel carefully using pipettes.
Run the sample till it travels the half area of the gel.
Removed the gel from the electrophoresis tank and observe it on UV transilluminator.

3.2.2.8 Spectrophotometric quantification of DNA

Spectrophotometry is the quantitative measurement of the reflection or transmission
properties of a material as a function of wavelength. spectrophotometry deals
with visible light,

near-ultraviolet,

and

near-infrared.

spectrophotometer

is

a photometer that can measure intensity as a function of the light source wavelength. A
spectrophotometer is commonly used for the measurement of transmittance or
reflectance of solutions, transparent or opaque solids, such as polished glass, or gases.
However they can also be designed to measure the diffusivity on any of the listed light
ranges that usually cover around 200 nm - 2500 nm using different controls
and calibrations. Within these ranges of light, calibrations are needed on the machine
using standards that vary in type depending on the wavelength of the photometric
determination.
Procedure
23 | P a g e

 Switched ON the UV Double beam Spectrophotometer and put the type of DNA to be
quantified.
Prepare the dilution 10 l dsDNA sample and 990l ultrapure distilled water.
Then kept the sample in spectrophotometer and take the reading of sample at 260/280
nm.
Prepare a known dilution of DNA sample in TE buffer which was used to dissolve the
DNA sample.
Recorded the OD of the sample at 260 and 280 nm.
Calculated the concentration of the DNA from the observed reading of absorbency.
Absorption ratio=260/280 nm wavelength
A260/280=1.7-2.0 (1.8)
A-260-Absorption by DNA
A-280-Absorption by protein

3.2.2.9 Polymerase Chain Reaction

PCR is a rapid technique for in vitro amplification of a specific DNA fragment by use
of two short single stranded primer flanking this segment.PCR were used to amplify the
desired region of the gene.the PCR reaction was done in a sterile 0.5 ml tube,the
reaction mix was made in the following order.
3.2.2.9.1 Preapration of PCR Mixture
Table 3: Reagent for master mix preparation.
Chemical
PCR buffer
dNTP
primer
universal forward primer
24 | P a g e

Stock
10x
2.5mM
100ppm

Working
2l(1x)
2.5l(0.2mM/L)
1l(8ppm)

Reverse primer vibrio alginolyticus

Reverse primer vibrio vunificus
Reverse primer vibrio cholera
Reverse primer vibrio mimicus
Reverse primer vibrio parahaemolyticus
Taq DNA polymerase
Distilled water
DNA sample
Total

1l(8ppm)
1l(8ppm)
1l(8ppm)
1l(8ppm)
1l(8ppm)
1l
7.5l
5l
20l

Procedure

Mixed the reagent 0.5 ml tubes or the 0.2 ml PCR tubes.

Be sure to keep the reagent on ice .
Vortex the tube so that all the composition mix properly.
Placed all the tube inside the thermocycler.

3.2.2.9.2 PCR Condition

Table 4: Required condition for PCR amplification
Steps
Heating lid
Initial denaturation
Denaturation
Annealing
Extension
Final extension
No.. fo cycles

Temperature Required
105C
94C for 3 min
94C for 30sec
60C for 30 sec
72c for 1min
72c for 7min
35

Until completion of PCR,hold sample at 4C.prepare the DNA for loading by addition
of 1/10 volume stop-loading buffer(contain EDTA,glycerol and bromophenol blue).
Analyse by agarose gel electrophoresis and be sure to be include size marker in at least
one well on the same gel.
The amplification products were visualized after electrophoresis at 50V for 45 min on a
1.2% gel.

25 | P a g e

4. RESULT AND DISCUSSION

4.1. Experimental sample
We done this experiment by taking 26 different water sample from different area of
Lucknow region specially and some sample from other regions(Ajamgarh tap water sample
from Gorakhpur region,Yamuna and Hindun river water from Delhi,Ghagra river sample
from Barabanki).10 sample of tap water,6 sample pond water,5 sample of river water,5
sample of fish water.
The given sample collected for this study

fig 4.1.1- Tap water samples- (Left to Right) Ajamgarh,Jankipuram,Khurram Nagar,Gudamba

Thana,CytoGene,Bakshi Ka Talab,Fajulaganj,BBAU,Adil Nagar,Munshi Pulia.

26 | P a g e

fig 4.1.2-Pond water samples- (Left to Right)Dhatingra,Jankipuram,Bsawan Purwa,Raitha

pond, Raitha pond ,Bakshi Ka Talab.

fig 4.1.3-River water samples-(Left to Right) Gomti river,Yamuna river,Hindun river,Ganga

river,Ghagra river

Fig 4.1.4-Fish water samples-(Left toRright) Dhatingra,Jankipuram,Munshi pulia,Khurram

Nagar,Tadikhana.
4.2 Spreading of water sample on TCBS media
After collecting the samples spreading was done on TCBS media for checking the bacterial
presence in given tap water, pond water, river water and fish water. After collection of all
27 | P a g e

samples took 200l aliquots of each sample and spread it with glass spreader on each of the
labeled TCBS media. Incubated these plates at 30C for 72 hours.

4.2.1 Tap water spreading:

Fig 4.2.1.1- TCBS media

28 | P a g e

Fig4.2.1.2:TCBS media;Adil Nagar tapwater spreaded on TCBS media; yellow colonies

obtained;sucrose positive.
The yellow colonies are grown at corner wall of TCBS spreaded petriplate after incubation of 48
hours.The yellow colonies shows the sucrose positive.

Fig4.2.1.3:TCBS media; Ajamgarh tap water spreaded on TCBS media; no colonies obtained.
No colony grown in Ajamgarh water sample.

29 | P a g e

Fig4.2.1.4:TCBS media;Bakshi Ka Talab tap water spreaded on TCBS media; no colonies

obtained.
No colony grown in Bakshi Ka Talab water samples.

Fig4.2.1.5:TCBS media; BBAU tap water spreaded on TCBS media; Green colonies
obtained;sucrose negative.
The small dispersed green colonies are grown on TCBS spreaded petriplate after incubation of
48 hours.The green colonies shows the sucrose negative.

Fig4.2.1.6:TCBS media;CytoGene tapwater spreaded on TCBS media; yellow and green

colonies obtained;sucrose positive(Y) and sucrose negative(G).
30 | P a g e

Large yellow and green pointed colonies are grown on TCBS spreded petriplate after incubation
of 48 hours.The yellow colonies shows the sucrose positive and green shows sucrose negative.

Fig4.2.1.7:TCBS media;Fajulaganj tapwater spreaded on TCBS media; no colonies obtained.

No colony grown on Fajulaganj water sample.

Fig4.2.1.8:TCBS media;Gudamba Thana tap water spreaded on TCBS media; no colonies

obtained.
No colonies grown in Gudamba Ka Thana water sample.

31 | P a g e

Fig4.2.1.9:TCBS media;Jankipuram tapwater spreaded on TCBS media; Green colonies

obtained; sucrose negative.
Small spreaded green colonies are grown on TCBS spreaded petriplate after incubation of 48
hours.The green colonies shows the sucrose negative.

Fig4.2.1.10:TCBS media;CytoGene tapwater spreaded on TCBS media; yellow colonies

obtained;sucrose positive.
Large pointed yellow colonies are grown on TCBS spreded petriplate after incubation of 48
hours.The yellow colonies show the sucrose positive.

32 | P a g e

Fig4.2.1.11:TCBS media;Munshi Pulia tapwater spreaded on TCBS media; yellow and green
colonies obtained;sucrose positive(Y) and sucrose negative(G).
Small and large spreded form of green and yellow colonies grown on TCBS spreaded petriplate
after incubation of 48 hours.The yellow colonies show the sucrose positive and green show the
sucrose negative.
4.2.2 Pond water spreading:

Fig4.2.2.1:TCBS media;Bakshi Ka Talab pond water spreaded on TCBS media; Green colonies
obtained;sucrose negative.
Small and large green colonies grown on TCBS spread petriplate after incubation of 24 hours.
The green colonies show the sucrose negative.
33 | P a g e

Fig4.2.2.2:TCBS media;Bsawan Purwa pond water spreaded on TCBS media; yellow colonies
obtained;sucrose positive.
Small dispersed and large pointed yellow colonies grown on TCBS spread petriplate after 24
hours. The yellow colonies show the sucrose positive.

Fig4.2.2.3:TCBS media;Dhatingra pond water spreaded on TCBS media; yellow and green
colonies obtained;sucrose positive(Y) and sucrose negative(G).
Small green and yellow colonies are grown on TCBS spread petriplate after incubation of 24
hours. The yellow colonies show the sucrose positive and green show the sucrose negative.

34 | P a g e

Fig4.2.2.4:TCBS media;Jankipuram pond water spreaded on TCBS media; yellow colonies

obtained;sucrose positive.
Small dispersed yellow colonies are grown on TCBS spread petriplate after incubation of 24
hours. The yellow colonies show the sucrose positive.

Fig4.2.2.5:TCBS media;Raitha pond water spreaded on TCBS media; yellow colonies

obtained;sucrose positive.
Large and small dispersed yellow are grown on TCBS spread petriplate after incubation of 24
hours. The yellow colonies show the sucrose positive.

35 | P a g e

Fig4.2.2.6:TCBS media;Raitha pond water spreaded on TCBS media; yellow colonies

obtained;sucrose positive.
Large and small dispersed yellow are grown on TCBS spread petriplate after incubation of 24
hours. The yellow colonies show the sucrose positive.
4.2.3 River water spreading:

Fig4.2.3.1:TCBS media;Ganga river water spreaded on TCBS media; Green colonies obtained;
sucrose negative.
Small green colonies grown on TCBS spread petriplates after incubation of 24 hours.The green
colonies show the sucrose negative.
36 | P a g e

Fig4.2.3.2:TCBS media;Ghagra river water spreaded on TCBS media; yellow and Green
colonies obtained; sucrose positive and sucrose negative.
Small green and large yellow colonies are grown on TCBS spread petriplate after incubation of
24 hours. The yellow colonies show the sucrose positive and green show the sucrose negative.

Fig4.2.3.3:TCBS media;Gomti river water spreaded on TCBS media;Yellow and

Green

colonies obtained; sucrose positive(Y) and sucrose negative(G).

Large,small yellow and dispersed green colonies grown on TCBS media spread petriplate after
incubation of 24 hours. The yellow colonies show the sucrose positive and green show the
sucrose negative.

37 | P a g e

Fig4.2.3.4:TCBS media;Yamuna river water spreaded on TCBS media; Yellow colonies

obtained; sucrose positive.
Small yellow dispersed colonies are grown on TCBS media spread petriplates after incubation
of 24 hours. The yellow colonies show the sucrose positive.

Fig4.2.3.5:TCBS media;Hindun river water spreaded on TCBS media; Yellow colonies

obtained; sucrose positive.
Large yellow colonies are grown on TCBS media spread petriplates after incubation of 24 hours.
The yellow colonies show the sucrose positive.

38 | P a g e

4.2.4 Fish water spreading:

Fig4.2.4.1:TCBS media;Dhatingra fish water spreaded on TCBS media; yellow colonies

obtained; sucrose positive.
Large,Small dispersed yellow colonies are grown on TCBS media spread petriplates after
incubation of 24 hours. The yellow colonies show the sucrose positive.

Fig4.2.4.2:TCBS media;Jankipuram fish water spreaded on TCBS media; yellow colonies

obtained; sucrose positive.
Large and Small yellow colonies are grown on TCBS media spread petriplates after incubation
of 24 hours. The yellow colonies show the sucrose positive.

39 | P a g e

Fig4.2.4.3:TCBS media;Khurram Nagar fish water spreaded on TCBS media; yellow colonies
obtained; sucrose positive.
Small yellow colonies are grown on TCBS media spread petriplates after incubation of 24 hours.
The yellow colonies show the sucrose positive.

Fig4.2.4.4:TCBS media;Munshi Pulia fish water spreaded on TCBS media;Yellow and Green
colonies obtained; sucrose positive(Y) and sucrose negative(G).
Small,large green and small,large yellow colonies are grown on TCBS media spread petriplate
after 24 hours. The yellow colonies show the sucrose positive and green show the sucrose
negative.
40 | P a g e

Fig4.2.4.5:TCBS media;Tadikhana fish water spreaded on TCBS media;Yellow and Green

colonies obtained; sucrose positive(Y) and negativ(G).
The yellow and green colonies are grown on TCBS media spread petriplates after incubation
of24 hours.The yellow colonies shows the sucrose positive growth.The green colonies shows
the sucrose negative growth.
4.3 Streaking:
For taking pure single colony from the TCBS media streaking was done on TSA media.

Fig4.3.1:TSA media;jankipuram and Khurram Nagar tap water streaking.

41 | P a g e

Fig 4.3.2:TSA media;cytogene tap water streaking

Fig 4.3.3:TSA media;Adil Nagar,BBAU,Munshi Pulia tap water (Green and yellow) colony
streaking.

Fig 4.3.4:TSA media;Jnkipuram fish water(Y),Bakshi Ka Talab(G) pond water,Raitha pond

water(Y),Gomti river(Y) streaking.
42 | P a g e

Fig 4.3.5:TSA media;Bsawan Purwa pond water(Y),Dhatingra pond water(Y),Raitha pond

water(Y),jankipuram pond water(Y) streaking.

Fig 4.3.6:TSA media:Munshi Pulia fish water(Y &G),Gomti river(G),Dhatingra fish water(Y)
streaking.

Fig 4.3.7:TSA media;Hindun river(Y),Yamuna river(Y),Ghagra river(G & Y) streaking.

43 | P a g e

Fig 4.3.8:TSA media;Dhatingra pond water(G) streaking.

Fig 4.3.9:TSA media;Tadikhana fish water(G & Y),Khurram nagar fish(Y) water
streaking.

Fig 4.3.10:TSA media;Hindun river(Y),Yamuna river(Y),Ghagra river(G & Y) streaking.

44 | P a g e

4.4 Broth culture:

For DNA isolation we required a liquid medium of bacteria so that i created a liquid
suspension culture of bacteria(TS broth).

Fig 4.4.1: TS Broth inoculated from TSA media(left to right)-BBAU tap water(G),Adil Nagar
tap water(y),Munshi Pulia tap water(G & Y),Dhatingra pond water(G & Y),Jankipuram pond
water(Y),Bsawan Purwa(Y),Raitha pond water (y)

Fig 4.4.2: TS Broth inoculated from TSA media(left to right)-Ghagra River(G & Y),Hindun
River(Y),Yamuna River(Y),Khurram Nagar fish water(Y),Tadikhana fish water(Y),Bakshi Ka
Talab Pond water(G),Gomti River(G & Y),Dhatingra fish water(Y),Jankipuram fish water(Y).

45 | P a g e

Fig 4.4.3: TS Broth inoculated from TSA media(left to right)-Munshhi Pulia fish water(G &
Y),Tadikhana fish water(G),Ganga river(G).

Fig 4.4.4: TS Broth inoculated from TSA media(left to right)-Jnakipuram tap water(G),Khurram
Nagar tap water(Y),CytoGene tap water(G),CytoGene tap water(Y).
4.5 DNA isolation

Fig 4.5.1 :(Left to Right)Isolated DNA and reagent used in DNA isolation-1X TE buffer,10%
SDS, phenol:chloroform:isoamyl alcohol,Propanol.
46 | P a g e

4.6 Agarose Gel Electrophoresis(0.8%)

For the confirmation of DNA presence 0.8 % gel was prepared in order to visulaize DNA under
UV transilluminator.

Fig 4.6.1: Reagent used for Agarose gel formation-1X TAE buffer and Agarose.

1
8

2
9

10

Fig 4.6.2:- 0.8% Agarose gel visualized in Gel documentation System. Lane 1: ladder 2:
Jankipuram 3:Khurram nagar 4:Polytechnic chauraha 5: BBAU 6:Adil nagar 7:Munshi
pulia 8:Dhatingra pond water 9:Jankipuram pond water: Samples of DNA isolated TSA broth

47 | P a g e

1
6

Fig 4.6.3:- 0.8% Agarose gel visualized in Gel documentation System. Lane 1: ladder
2:Bsawan purwa 3:Raitha pond

4:Bakshi ka talab 5:Gomti river: Samples of DNA isolated

TSA broth
4.7 Spectrophotometric quantification of DNA using UV double beam spectrophotometer:
The quality of DNA was estimated by measuring the 260:280 UV absorbance ratios which
varied between 1.6 and 1.8.following table shows the value obtained for each sample when
analyzed at 260 and 280 nm.
4.7.1 Tap water
Table 5: Spectrophotometric analysis of DNA (tap water)
S.No
1
2
3
4
5
6
7
8

Tap water samples

Jankipuram
Khurram Nagar
CytoGene G
CytoGene y
BBAU
Adil Nagar
Munshi Pulia y
Munshi Pulia y

48 | P a g e

M factor
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00

Ratio
1.03
1.02
1.04
1.02
0.96
1.01
1.02
1.01

DNA Conc
1.53
4.86
5.87
5.56
1.35
5.91
5.42
4.07

Protein Conc
3.41
11.06
12.80
12.70
3.41
13.74
12.34
9.47

4.7.2 Pond water:

Table 6: Spectrophotometric analysis of DNA (Pond water)
S.No

Pond water samples

M factor

Ratio

DNA Conc

Protein Conc

Dhatingra G

1.00

1.02

2.80

6.37

Dhatingra Y

1.00

1.01

1.88

4.36

Jankipuram Y

1.00

1.06

7.03

14.82

Bsawan Purwa

1.00

0.97

0.93

2.31

Raitha Pond

1.00

1.03

4.03

9.05

Raitha Pond

1.00

1.02

2.20

5.01

Bakshi Ka Talab

1.00

1.02

4.25

9.66

4.7.3 River Water

Table 7: Spectrophotometric analysis of DNA (river water)
S.No

River

water M factor

1
2
3
4
5
6
7

samples
Gomti G
Gomti Y
Yamuna
Hindun
Ganga
Ghangra G
Ghangra Y

1.00
1.00
1.00
1.00
1.00
1.00
1.00

Ratio

DNA Conc

Protein Conc

1.09
1.04
1.04
0.99
0.99
1.00
1.00

6.61
5.35
5.89
1.92
2.07
1.53
1.67

13.22
11.69
13.01
4.56
4.98
3.58
3.90

4.7.4 Fish Water

Table 8: Spectrophotometric analysis of DNA(fish water)

49 | P a g e

S.No

Fish

water M factor

1
2
3
4
5
6
7

samples
Dhatingra
Jankipuram
Munshi Pulia G
Munshi Pulia Y
Khurram Nagar
Tadikhana G
Tadikhana Y

1.00
1.00
1.00
1.00
1.00
1.00
1.00

Ratio

DNA Conc

Protein Conc

1.02
1.02
1.02
1.02
1.02
0.98
1.01

2.78
2.22
2.92
1.38
2.24
0.57
1.97

5.27
5.02
5.57
2.97
5.08
1.39
4.55

4.8 Multiplex PCR

For confirmation of bacteria which is resposible for contaminating each water sample using
3 primers with each bacteria. The bacteria which give yellow colour on TCBS media those
DNA amplified with vibrio alginolyticus, vibrio vulnificus, Vibrio cholera. And those who
give green colour on TCBS those DNA amplified with vibrio vulnificus, vibrio mimicus and
vibrio parahaemolyticus.
4.9 Agarose gel electrophoresis (1.2%)
The amplified DNA run on 1.2% for the confirmation of which primer bind to the bacterial
DNA.

1
8

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2
9

3
10

Fig 4.9.1 Amplified DNA using specific primers run on a 1.2 % Agarose gel
lane 1: marker; lane 4-Jankipuram tap water(V.P),lane 7-Munshi pulia tap
water(V.P),lane 8-Adil nagar tap water(V.V).

1
5

2
6

3
7

Fig 4.9.2 Amplified DNA using specific primers run on a 1.2 % Agarose gel
lane 1: marker; lane 2-Jankipuram pond water(V.V),lane 4-Raitha pond
water(V.C),lane 7-Bsawan purwa pond water(V.A)

51 | P a g e

1
8

2
9

3
10

Fig 4.9.3 Amplified DNA using specific primers run on a 1.2 % Agarose gel
lane 1: marker; lane 2:Gomti river(V.P,V.M),lane 4-Yamuna river(V.V),lane 5Ghagra river(V.P)

1
5

2
6

Fig 4.9.4 Amplified DNA using specific primers run on a 1.2 % Agarose gel
lane 2: marker; lane 2-Dhatingra fish water(V.A),lane 3-Munshi pulia fish
water(V.V).

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1
6

2
7

Fig 4.9.5 Amplified DNA using specific primers run on a 1.2 % Agarose gel
lane 2: marker; lane 4-Khuram nagar fish water(V.V),lane 7-Tadikhana fish
water(V.P)

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4.10 Table of vibrio species present in given water samples:

Table 9: Amplification observed in different water samples with V.cholerae, V.parahaemolyticus,
V.vulnificus, V.alginolyticus, V.mimicus
S.NO
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.

WATER SAMPLES

COLON

Ajamgarh tap water

Jankipuram tap water
Khurram nagar tap water
Gudamba thana tap water
Cytogene tap water
Bakshi ka talab tap water
Fajulaganj tap water
BBAU tap water
Adil nagar tap water
Munshi pulia tap water
Dhatingra pond water
Jankipuram pond water
Bsawan purwa pond water
Raitha pond 1 pond water
Raitha pond 2 pond water
Bakshi ka talab pond water
Gomti river
Yamuna river
Hindun river
Ganga river
Ghagra river
Dhatingra fish water
Jankipuram fish water
Munshi pulia fish water
Khurram nagar fish water
Tadikhana fish water

Y
-------G
Y
--------G,Y
----------------G
Y
G,Y
G,Y
Y
Y
Y
Y
G
G,Y
Y
Y
G
G,Y
Y
Y
G,Y
Y
G,Y

V.A

V.V

(yellow)

(green)

V.C

V.M

V.P

Yellow colony bacteria = V.A, V.V, V.C

Green colony bacteria =V.V, V.M, V.P
Vibrio alginolyticus = yellow colony
Vibrio vulnificus = green 85% or yellow 15% colony

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V.V

Vibrio cholera =yellow colony

Vbrio mimicus = green colony
Vibrio parahaemolyticus = green centered colony

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5. CONCLUSION
The samples were collected in order to detect presence of human pathogenic vibrio species in
water bodies and the presence of vibrio species was confirmed by spreading water sample on
TCBS media, incubated for 48 hours at 37C resulting in many different colonies. It was
subjected to streaking on TSA media and incubated it for 24 hours (37C) followed by
inoculating it in TS broth, incubated for 24 hours (37C). DNA was isolated from each sample
by phenol:chloroform method and the presence of specific vibrio species in water samples
detected by Multiplex PCR amplification method with different primers of vibrio species
(V.cholerae, V.parahaemolyticus, V.vulnificus, V.alginolyticus, V.mimicus).The Amplified
product was analysed by electrophoresis, it shows the presence of particular human pathogenic
vibrio species in water samples. Among the tap water sample railway station (Ajamgarh),
Gudamba Thana (Lucknow), Bakshi ka Talab (Lucknow), Fajulaganj (Lucknow) were free from
vibrio as absence of amplified band showed but the sample of Jankipuram (Lucknow) showed
the presence of vibrio parahaemolyticus, sample of Khurram Nagar showed the presence of
vibrio vulnificus and vibrio cholera, sample of Polytechnic chauraha showed the presence of
vibrio vulnificus, sample of BBAU showed the presence of vibrio mimicus, sample of Adil
Nagar showed the presence of vibrio vulnificus and sample of Munshi Pulia showed the
presence of vibrio parahaemolyticus. Among the pond water sample Dhatingra showed the
presence of V.vulnificus& V. parahaemolyticus, sample of Jankipuram showed the presence of
V.vulnificus, sample of Bsawan Purwa showed the presence of V. alginolyticus,sample of Bakshi
ka Talab showed the presence of V.parahaemolyticus,sample of Raitha pond 1st and 2nd water
showed the presence of V.cholerae.Among the River water sample Gomti river showed the
presence of vibrio vulnificus, cholera & parahaemolyticus, sample of Yamuna river showed the
presence of vibrio vulnificus, sample of Hindun river showed the presence of vibrio
alginolyticus & vibrio vulnificus, sample of Ghagra showed the presence of vibrio
parahaemolyticus. All samples showed the presence of specific primers except Ganga River.
Among the Fish water sample Dhatingra fish water showed the presence of V.alginolyticus &
V.cholerae,

sample

of

Munshi

Pulia

showed

the

presence

of

V.vulnificus

&

V.parahaemolyticus,sample of Khurram Nagar showed the presence of V.cholerae,sample of

Tadikhana fish water showed the presence of V.parahaemolyticus. All fish water showed the
presence of vibrio species except Jankipuram fish water.
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