1

Diagnosis of NEISSERIA GONORRHOEAE using molecular beacon.

Divya Sachdev (Ph.D)1, Achchhe Lal Patel (Ph.D)1 Subash Chandra Sonkar (M.Sc.)1, Indu

Kumari (M.Sc.)1, Daman Saluja (Ph.D)1#

Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110007.

Email-id: dsalujach59@gmail.com.

Address for Reprint: Same as corresponding author.

Other Information

Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi-110007.
Corresponding author: Daman Saluja

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Number of text pages: 17

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Number of Tables: 3

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Number of Figures: 2

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Funding :

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The work was supported by core funding from Department of Biotechnology, Ministry of

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Science and technology, India .

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Conflict of interest

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The authors have no commercial or other association that might pose a conflict of interest.

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Key words

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Molecular beacon, Point of care test, Neisseria gonorrhoeae, dry swabs.

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Supplementary Figure:2

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Abstract

22

Neisseria gonorrhoeae is an important STI causing pathogen worldwide. Due to absence of an

23

affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in

24

developing countries where infection rates are maximum. Treatment is given on the basis of

25

symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive,

26

specific, PCR based visual diagnostic assay suitable for developing countries, required for better

27

disease management is aimed in present study. Endocervical swabs were collected from patients

28

visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was

29

developed and modified to visual assay using molecular beacon for end-point detection. It was

30

evaluated against Roche Amplicor NG kit and rmp gene. Specificity of beacon was confirmed by

31

competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas

32

negative and positive predicted value were 99.40% and 98.78% respectively. We also observed

33

that twice the concentration (2X) of pre-mix was stable at 4C for 4 months and dry swab

34

samples gave concordant results with that of wet swabs. These features make the test best

35

suitable for routine diagnosis of genital infections in developing countries.

36
37

INTRODUCTION

38

Neisseria is one of the most common STD causing pathogen. WHO estimated that 106 million

39

cases occur annually with approximately 2/3rd of these cases reported from developing countries

40

[1]. Although easily curable by a single dose of antibiotic, large population goes undiagnosed

41

due to absence of rapid, sensitive, specific and cost effective diagnostic methods. Early diagnosis

42

and treatment can also reduce the risk of the patient developing long-term complications of the

43

disease. The available diagnostic methods like gram stain and culture are economical but have

44

poor sensitivity especially in asymptomatic patients. Moreover, delay in availability of the results

45

not only makes these methods unsuitable for screening, but also unsuitable for treatment as a

46

number of patients (especially adolescents and pregnant women) do not return to collect their

47

results [2, 3]. NAAT based commercial kits are also available for diagnosis of Neisseria but due

48

to the high cost, these do not meet the requirement of mainstream laboratories in developing

49

countries where majority of world population is afflicted. Syndromic management introduced by

50

WHO not only lacks both sensitivity (30% - 80%) and specificity (40% - 80%) for the infections

51

in women with vaginal discharge [2, 4, 5] and also misses asymptomatic patients [6, 7]. A

52

recently introduced POC test, PATH GC check rapid test (program for Appropriate technology

53

Seattle, USA) is an immunochromatographic strip test, with moderate sensitivity ranging from

54

81% to 31.2% under different clinical setting as compared to Roche Amplicor CT/NG PCR

55

assay and 16s rRNA PCR assay [8]. Highly sensitive realtime PCR kits recently approved for in-

56

vitro diagnosis are exceedingly expensive and out of reach of even best clinical laboratories in

57

the developing world. Therefore, there is an urgent need for a sensitive, rapid, cost effective,

58

point-of-care test in developing countries for routine detection of Neisseria so as to prevent the

59

widespread infection and development of reproductive sequale.

60

In the present study, we have modified the previously developed PCR assay for the detection of

61

Neisseria [9], so as to make it more specific, sensitive and easy to use. By using molecular

62

beacon for end-point detection, the assay eliminates agarose gel electrophoresis to visualize the

63

amplicon, increases specificity of assay by hybridizing to the internal sequence of the amplicon

64

and minimizes the risk of cross contamination and carry over contamination to other clinical

65

samples [10]. Further, we have stabilized the PCR mix at 4C for upto 4 months as well as

66

standardized the use of dry swabs for collection of samples in empty vials, which can easily be

67

transported at room temperature (upto 30C) making it a method of choice in peripheral

68

laboratories with minimum resources [11]. In brief, the molecular beacon based PCR assay

69

described here meets the essential requirements for use in resource limited settings.

70
71

2. Materials and methods

72

2.1 Patient population

73

Endocervical swab samples from non-pregnant women (18 years 55 years of age with 90%

74

patients in the age group of 18 - 31 years) seeking diagnosis and treatment of vaginal discharge

75

syndrome (VDS) or PID, were collected during 2008 - 2009 and 2011 - 2012 from the outpatient

76

departments of gynaecology from various hospitals Delhi as per the guidelines of Indian Council

77

Of Medical Research, India and adopted by Institutional Ethical committee of Dr. B.R.

78

Ambedkar

79

Eth.Com/ACBR/11/2107). Patients on antibiotic treatment were excluded from the study.

80

Written informed consent was taken from all enrolled in the study patients.

81

2.2 Specimen collection and processing

82

After thorough speculum examination of vulva for warts, ectopy and discharge by the attending

83

clinician, two endocervical swabs were collected from each patient. Our study was divided into

84

two groups. For group 1 (n = 412), the first swab was placed in a vial containing AMPLICOR

85

Specimen Transport Medium and the second swab was placed in 1 ml transport medium

86

(Phosphate Buffer Saline with 1mM EDTA) [12, 13]. For group 2 (n = 133), the first swab was

centre

for

Biomedical

Research,

University

Of

Delhi,

(No.F50-2/

87

placed in transport medium and second swab was placed in the empty vial (dry swab). All the

88

wet swab specimens were transported same day to microbiology laboratory on ice while the dry

89

swabs were transported at room temperature (25C - 30C) and tested either on the same day or

90

stored at subzero temperature till further use. Dry swabs were immersed in PBS (1ml) for 10 min

91

before processing. All samples were processed by enzymatic lysis method as described

92

previously [13, 14].

93

2.3 Primers and probes used

94

The primers used in our previous studies designed against the orf1 gene sequences [9] were

95

modified to improve specificity and sensitivity of the assay. The modified primers (orf1F 5-

96

GATCCAACTATTCCCGATTGC-3 and orf1R 5- GCAAAGTTATACAGCTTCGCCTGA-

97

3) for orf1 gene of N. gonorrhoeae produced an amplicon of size 269 bp. Molecular beacon, 5-

98

Fluorophore -CCATGCG TGACTGCCAACAAGAAAAAAGCCATCC CGCATGG-Quencher-

99

3 (Eurogenetic, Belgium; Tm = 69.7C) complementary to region within amplicons of orf1

100

gene, was designed as described by Tyagi and Kramer [15] with melting temperature of beacon

101

5-7C higher than annealing temperature of primers. Quencher flourophore pair was chosen such

102

that emission spectrum of fluorophore overlapped with absorption spectrum of quencher. Two

103

different fluorophore - quencher pairs: Cy3 - Dabcyl and FAM - BHQ2 were used.

104

2.4 PCR Amplification

105

Supernatant (5l) of processed sample or crude lysate was used for PCR [13, 14]. The PCR mix

106

contained 50 mM KCl, 10 mM Tris-Cl pH 8.3, 2.0 mM MgCl2), 200 M each of the four dNTPs

107

(New England Biolabs Inc.), 10 pmoles each of forward and reverse primers, 1U of Taq DNA

108

polymerase (Bangalore Genei India Pvt. Ltd., India). Negative (No template) and positive

109

controls (orf1 gene cloned in TA vector) were included in each PCR run. Amplification of orf1

110

gene was performed in thermal cycler (Bio-Rad) for 35 cycles with following parameters: 95C

111

for 5 min for initial denaturation, cycling of 95C for 30 sec, 60C for 30 sec, 72C for 30 sec,

112

final extension at 72C for 10 min. The amplicons were analyzed by agarose gel (1%)

113

electrophoresis (Figure S1).

114

The amplicons from positive samples (10% of the total) were eluted from agarose gel using

115

DNA isolation kit (Geneaid, USA) according to the manufacturers instructions and sequenced

116

using PCR primers. DNA sequence of the amplified product was compared to the known orf1

117

nucleotide sequences (AE004969.1, NGO0365) in the GenBank databases using BLAST

118

program to determine the percent identity.

119

2.5 Roche AMPLICOR MWP Neisseria gonorrhoeae Detection Assay

120

412 endocervical specimens (group I) were tested by Roche AMPLCOR Multi-well plate NG

121

detection kit (Roche Diagnostic Systems) according to the manufacturers instructions.

122

2.6 Use of molecular beacons

123

Asymmetric PCR was standardised using 40 pmoles of sense primer and 20 pmoles of antisense

124

primer amplifying more copies of strand complementary to probe [16]. Complementary strands

125

generally anneal at higher temperature than molecular beacon hybridising to its target during

126

ramping. Asymmetric PCR leaves higher amount of target strand for beacon to hybridize. This

127

reduces the competition of beacon with its target and thus helps in obtaining better signal and

128

minimum noise [16]. The amplified product and beacon mix was again heated to 95C for 5

129

minutes and was slowly cooled to 20C at ramp rate of 0.1C/second. The temperature cycle

130

added to PCR cycling conditions leads to maximum binding of beacon to its target sequence.

131

Unbound probe hybridises back to hair pin shape as temperature slowly cools below its melting

132

temperature thus reducing the noise. The products were checked by i) Fluorescent ELISA Reader

133

ii) Dark Reader and iii) Agarose gel electrophoresis. To visualize the amplicons using ELISA

134

reader, the reaction contents (50 l) were transferred into 96 well plate. To each well, 150 l of

135

20 mM Tris/Cl (pH 7.4) was added and the fluorescence was measured using appropriate

136

excitation/emission wavelength pair [17] using a spectrofluorimeter (M200 infinite, Tecan Group

137

Ltd.) at 37C. Clinical Samples (n = 412) were tested using Fam-BHQ2 pair and randomly

138

selected clinical samples (n = 80) were tested using Cy3-Dabcyl pair. The excitation wavelength

139

of Fam is 491 nm and emission wavelength 521 nm and excitation wavelength for cy3 is 554 nm

140

and emission wavelength is 568 nm. In method ii) the tubes were placed in the slot of in-house

141

dark reader manufactured by DSS Tech Pvt Ltd for detection of fluorescence by visual

142

inspection.

143

2.7 Evaluation of sensitivity and specificity of molecular beacon

144

Different concentrations of molecular beacon (0.2 pmoles to 1 pmoles) were used to standardise

145

the concentration with minimum noise and maximum signal. Further to evaluate the sensitivity

146

of molecular beacon, serial dilutions from 100 ng to 10 fg of purified Neisseria genomic DNA

147

were used. We have also used non-specific unlabeled probe in excess (specific: non-specific ratio

148

ranging from 1:1 to 1: 50) along with specific probe in PCR reactions. All assays were repeated

149

at least five times.

150

2.8 Stabilisation of PCR reaction mixture at 4C

151

Master mix containing all the reagents other than template in twice the concentration was stored

152

at 4C for different time intervals. An aliquot of master mix was checked at zero time for the

153

activity of the enzyme. Aliquots of master mix were checked at regular intervals till seven

154

months for the PCR assay.

155

2.9 Statistical analysis

156

All statistical analysis was performed using Graphpad prism 5 software (GraphPad Software,

157

Inc.).

158

3. Results

159

3.1 Clinical performance of the in-house PCR using modified primers

160

Primers published earlier were slightly modified for carrying out detection using molecular

161

beacon. Efficacy of in-house PCR for N. gonorrhoeae was evaluated against Roche Amplicor

162

MWP kit and rmp gene [18]. All samples were tested using modified primers, Roche Amplicor

163

MWP kit and published primers for rmp gene using swab samples collected from 412

164

symptomatic women patients with the median age of 24 years. A sample testing positive with

165

two or more methods was scored positive whereas sample testing negative with two or more

166

methods was considered to be negative to calculate the performance characteristics of the test.

167

The modified orf1 primers were found to be 97.02% sensitive and 99.18% specific (Table 2).

168

3.2 Clinical evaluation of molecular beacon

169

Optimal concentration of beacon was standardized at 0.8 pmoles which gave maximum signal and

170

minimum noise (Figure 1, Panel A) and beacon was found to sensitive till 100fg of gDNA (Figure 1,

171

Panel C). We observed that fluorescence decreased to 60% on addition of 40 folds of unlabeled

172

specific probe whereas it remained unchanged on addition of unlabeled non-specific probe

173

(Figure 1, Panel B). A total of 412 clinical samples were checked for presence of Neisseria using

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Fam labelled molecular beacon detected using ELISA reader as well as dark reader (Figure 1,

175

Panel D and E). The second pair of fluorophore-quencher (Cy3-Dabcyl) was also used for

176

detection of randomly selected clinical samples (n=80) which produced similar results (Figure

177

S2) suggesting that molecular beacon worked effectively independent of the fluorophore-

178

quencher pair. Out of 412 samples 154 tested positive for Neisseria gonorrhoeae using above

179

four detection methods. There were three samples which were positive by agarose gel

180

electrophoresis, molecular beacon and rmp gene whereas tested negative by Roche Amplicor kit

181

and were considered as true positive based on composite reference standard method. There were

182

four samples which tested negative by in-house PCR but were positive by molecular beacon

183

method as well as by Roche Amplicor kit and rmp gene PCR. Two samples were false positive

184

by in-house PCR as they tested negative by molecular beacon and by other two detection

185

methods used. Thus, use of molecular beacon enhanced the sensitivity of our test. Only one

186

sample falsely tested positive using molecular beacon (Table 1). Based on composite reference

187

standard method, use of molecular beacon enhanced the sensitivity of our test to 98.21% and

188

specificity to 99.21%. Our test showed accuracy of 99.01%. We conclude that use of molecular

189

beacons for detection of amplicons not only reduce time of detection but also increases the

190

sensitivity (due to fluorescence) and specificity (due to hybridization of the beacon) of the

191

diagnostic tests with practically no false positives or false negative results (Table 2).

192

3.3 Use of dry swabs for clinical evaluation

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Since it is easier to transport dry swab samples, we checked the performance of in-house PCR

194

using dry swabs (n=133) and found similar sensitivities of PCR assay to that of gDNA isolated

195

from wet swabs. Out of 133 samples, 33 were positive when total DNA was extracted from dry

196

or wet swabs while 96 were negative for Neisseria gonorrhoeae infection. Discordance in PCR

197

results was observed for 4 samples using dry and wet swabs. All samples were also analysed

198

using housekeeping gene, rmp (as shown in Table 3) which helped in resolving discrepant

199

results. We found good concordance of results with dry and wet swabs, thus use of dry swabs is

200

recommended as a preferred method for sample collection.

201

3.4 Standardisation of reagents at 4C

202

Further to qualify an assay as user friendly and affordable diagnostics, premix of the reagents

203

(buffer, dNTPs, primers, probe, taq DNA polymerase) was made at twice the concentration and

204

kept at 4C for upto 7 months. PCR amplification was carried out at regular intervals and we

205

found that pre mix was stable upto 4 months making it an easy assay to be carried out in

206

peripheral laboratories where deep freezers are not available (Figure 2).

207

4. Discussion

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Diagnosis by a clinician can sometimes be made on the basis of signs and symptoms of the disease

209

but accurate diagnosis requires a specific diagnostic test, often requiring access to a clinical

210

laboratory. A reasonably good and accurate diagnostic test, is therefore of paramount importance

211

in reducing the burden of infectious diseases. For the past two decades, nucleic acid based PCR

212

assays have been profusely published as diagnostic methods [12-14, 19, 20]. PCR methods are

213

highly specific and sensitive and less time consuming than culture method. Inspite of this, PCR-

214

based diagnostic assays have failed to penetrate the market in developing countries. To be useful,

215

diagnostic methods must be accurate, simple and affordable for the population for which they are

216

intended. Due to the high cost of PCR based diagnostic methods and lack of infrastructure and

217

expertise available in resource limited setups, developing countries are still using assays which

218

have low sensitivity and specificity. A conventional PCR based assay can result in false positives

219

and experimental variability. Automatic systems which combine nucleic acid extraction with high

220

throughput PCR amplification, although reduce problems associated with manual sample

221

processing and visualization are highly expensive (more than $140,000) and out of reach of even

222

best clinical laboratories in the developing world. Most of these tests use real time techniques

223

which are not only expensive but also require technical expertise and are time consuming. The

224

present study was focussed on designing of an easy visualization based method for the detection

225

of Neisseria gonorrhoeae using molecular beacon. Our in-house PCR test is not only as sensitive

226

as that of CT/NG Roche Amplicor Multiwell plate kit but use of molecular beacon has made

227

detection far easier. In our test, DNA amplification was performed in routine thermal cycler

228

followed by end-point detection using molecular beacon on dark reader or ELISA reader. The

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amplicons can be detected by hybridization to fluorescent tagged molecular beacon and tubes

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can be visualised directly under an indigenous dark reader (costing around $1000). Since in most

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of the diagnostic laboratories, presence or absence of pathogen is required for deciding

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treatment, the end point detection by dark reader is adequate. Visualising the PCR products

233

directly in the PCR tube decreases the time of detection and also minimizes the chances of cross

234

contamination as well as carry over contamination which is highly beneficial to the user.

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In many developing countries, protocols for waste disposal and biosafety are either not

236

developed or are highly rudimentary. Containment of bio-hazardous material such as ethidium

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bromide etc is almost nonexistent [20]. The diagnostic assay described in this study, eliminates

238

the running of agarose gel which is time consuming, cumbersome and uses carcinogenic

239

chemicals like EtBr. Collection of dry swabs for clinical samples at ambient temperature (25C -

240

30C) makes it easy to maintain the temperature during transportation of samples from field to

241

nearby laboratories which further helps in reduction of cost spend for sample collection as well

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as transportation. Similarly, use of PCR reaction premix (containing PCR buffer, primers,

243

molecular beacon as well as Taq polymerase) reduces the need for training end user of

244

micropipetting. The use of premix will also eliminate the chance of contamination, and improves

245

consistency and repeatability by even untrained worker. The premix is also stable for at least four

246

months at 4C. Since it easier to maintain 4C in fridges (also during electricity failure one can

247

use cool packs, a situation quite common in resource poor settings and in rural areas) it is of

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great benefit for peripheral laboratories with minimal resources. Use of molecular beacon, dry

249

swabs for sample collection and use of dark reader to visualise results is expected to decrease the

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cost of test tremendously. The patients using Roche Amplicor kit based test for diagnosis of

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Neisseria are charged Rs 1500-2000 in different laboratories in India whereas our laboratory cost

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of our test is Rs 60 only. This amount falls in affordable range of large percentage of population

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of India and other developing countries thus helping in better diagnosis and efficient treatment

254

and disease management. In nutshell, the molecular beacon based PCR assay developed in the

255

present study address to a large extent the need of a simple cost effective, easy to use, highly

256

sensitive and specific nucleic acid amplification assay, a step forward towards developing point-

257

of-care diagnostics for low resource settings [21, 22]. To the best of our knowledge, this is the

258

first report of use of molecular beacons for diagnosis of Neisseria gonorrhoeae. Future work is

259

in progress to address the major challenge to automate the DNA extraction and PCR assay at an

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affordable price. Local production of such diagnostic kits will further reduce the cost. Faster,

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simpler and cost effective diagnostics will make disease control efforts more effective especially

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in places where patients have difficulty in reaching health care.

263

Refrences

264

1.World Health Organization. Global action plan to control the spread and impact of

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antimicrobial resistance in Neisseria gonorrhoeae WHO organization. 2012.

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2. Mukenge L, Alary M, Lowndes CM. Syndromic versus laboratory-based diagnosis of cervical

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infections among female sex workers in Benin: implications of non-attendance for return visits.

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Sex Transm Dis. 2002; 29:324-330.

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3. Schwebke J, Sadler R, Sutton J, et.al. Positive screening tests forgonorrhea and chlamydial

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infection fail to lead consistently to treatment of patients attending a sexually transmitted disease

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clinic. Sex Transm Dis. 1997; 24:181-184.

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4. Alary M, Lowndes CM, Mukenge-Tshibaka L, et.al. Sexually transmitted infections in male

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clients of female sex workers in Benin: risk factors and reassessment of the leucocyte esterase

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dipstick for screening of urethral infections. Sex Transm Infect. 2003. 79:388-392.

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5. Mayaud P, ka-Gina G, Cornelissen J, et.al. Validation of a WHO algorithm with risk

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6. Choudhry S, Ramachandran VG, Das S, et.al. Pattern of sexually transmitted infections and

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sexually transmitted infection clinic of a tertiary care hospital. Indian J Sex Transm Dis. 2010;

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31(2):104-108.

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7. Vishwanath S, Talwar V, Prasad R, et.al. Syndromic management of vaginal discharge among

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women in a reproductive health clinic in India Sex Transm Infect. 2000; 76:303-306.

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8. Alary M, Gbenafa-Agossa C, Aina G, et.al. Evaluation of a rapid point-of-care test for the

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detection of gonococcal infection among female sex workers in Benin. Sex Transm Infect. 2006

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9. Chaudhry U, Saluja D. Detection of Neisseria gonorrhoeae by PCR using orf1 gene as target.

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Sex Trans Infect. 2002; 78:72-78.

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10. Tsourkas A, Bao G. Shedding light on health and disease using molecular beacons. Brief

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Funct Genomic Proteomic. 2003; 1:372-384.

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11. Feng Q, Cherne S, Winer RL, et.al. Evaluation of transported dry and wet cervical exfoliated

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samples for detection of human papillomavirus infection. J Clin Microbiol. 2010; 48(9):3068-

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12. Chaudhry U, Ray K, Bala M, et.al. Multiplex polymerase chain reaction assay for the

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detection of Neisseria gonorrhoeae in urogenital specimens. Current Science. 2002; 83(5):634-

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13. Sachdeva P, Patel AL, Sachdev D, et.al. Comparison of an in-house PCR assay, direct

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fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C.

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trachomatis. J Med Microbiol. 2009; 58(Pt 7):867-873.

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14. Patel AL, Sachdev D, Nagpal P, et.al. Prevalence of Chlamydia infection among women

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visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection

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of Chlamydia trachomatis. Ann Clin Microbiol Antimicrob. 2010; 8:9:24.

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15. Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat

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Biotechnol. 1996; 14:303-308.

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16. Poddar SK. Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a

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target gene of adenovirus. Molecular and Cellular Probes. 2000; 14:25-32.

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17. Haldar S, Chakravorty S, Bhalla M, et.al. Simplified detection of Mycobacterium

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tuberculosis in sputum using smear microscopy and PCR with molecular beacons. J Med

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Microbiol. 2007; 56(Pt 10):1356-1362.

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18. Liebling MR, Arkfeld DG, Michelini GA, et.al. Identification of Neisseria gonorrhoeae in

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synovial fluid using the polymerase chain reaction. Arthritis Rheum. 1994; 37(5):702-709.

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19. Wolk D, Mitchell S, Patel R. Principles of molecular microbiology testing methods. Infect

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Dis Clin North Am. 2001; 15(4):1157-1204.

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20. Yager P, Domingo GJ, Gerdes J. Point-of-care diagnostics for global health. Annu Rev

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Biomed Eng. 2008; 10:107-144.

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21. Mabey D, Peeling RW, Ustianowski A, et.al. Diagnostics for the developing world. Nat Rev

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Microbiol. 2004; 2(3):231-240.

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22. Peeling RW, Holmes KK, Mabey D, et.al Rapid tests for sexually transmitted infections

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(STIs): the way forward. Sex Transm Infect. 2006; 82 Suppl 5:v1-v6.

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321

Acknowledgement

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Fellowship from CSIR (DS) and UGC (SCC, IK) and financial support by DBT is greatly

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acknowledged. We are grateful to all the patients and clinicians for their cooperation.

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Figures with Legends

328
329

Figure 1: Standardisation of use of moecular beacon for detection of N. gonorrhoeae: Panel

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A) Standardization of concentration of beacon Panel B) Specificity of Molecular Beacons using

331

unlabeled specific and non specific probe (1:0, 1:1, 1:5, 1:10, 1:20 and 1:50). Panel C)

332

Sensitivity of molecular beacon using purified Neisserial genomic DNA (100fg to 10ng). Panel

333

D) Detection of clinical samples in ELISA reader. The amplified PCR products were transferred

334

into wells of a 96-well plate containing 150 l of 10 mM Tris/Cl (pH 8.0) and the fluorescence

335

was measured using 492 nm excitation and 521 nm emission in a ELISA Reader. Panel E)

336

Direct visualization of PCR tube under dark reader. Tube 1=NTC, tube 2 = PTC, tube 3 =

337

clinical sample (positive).

338

339
340

Figure 2: Agarose gel showing the stability of PCR master mix at 4C: Amplification of orf1

341

gene was performed with PCR master mix stored at 4C for 0, 1, 2, 3, 4, 5, 6 and 7 months

342

(Lanes 2 - 9 respectively). Lane 1 is No template control. Lane M is 100 bp DNA ladder. Master

343

mix is found to be stable and functional for four months.

344
345
346
347

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349
350
351
352

TABLES

No. of samples

PCR

Beacon

Roche

rmp

Conclusion

154

Positive

Positive

Negative

Negative

Positive

Negative

Positive

Positive

Positive

Negative

237

Negative

353
354

Table 1: Comparison of use of molecular beacon as detection method with gel

355

electrophoresis, Roche Amplicor MWP kit and PCR with a house keeping gene-rmp.

356
357
358

PCR

Result of

Beacon

Roche Amplicor

Rmp

MWP kit

samples
(n = 412)

NG

Positive

Negative

Positive Negative

Positive

Negative

Positive Negative

163

165

165

164

242

243

242

242

97.02

93.19-

98.21

94.86-

98.21

94.86-

97.62

94.01-

positive
NG
negative
Sensitivity

99.02

(95%CI)
Specificity

99.18

98.79

97.98

99.40

95.34-

118.37

29.76-

98.78

0.03

98.3

Accuracy

0.01-0.07

96.68-

96.47-

239.64

33.89-

98.80

99

0.01-0.06

99.81

95.73-

98.78

96.46-

98.8

30.13-

98.37

98.7

0.01-0.06

95.8999.55

119

476.46
0.02

95.7199.82

99.73
119.82

97.0699.88

99.80

1694.66
0.02

97.06-

99.33

99.88

99.73

470.74
LR (-)

99.78

99.90

99.33

CI)
LR (+)

95.68-

97.73-

99.61

99.93

99.82

CI)
NPV (95%

99.59

99.88

(95%CI)
PPV (95%

97.06-

99.61

29.95473.60

0.02
98.5

0.01-0.06

359
360

Table 2: Comparison of sensitivity, specificity, positive predictive value (PPV), negative

361

predictive value (NPV), likelihood ratios (LR) and accuracy of different methods calculated with

362

95% confidence intervals (CI).

363

364

Clinical
samples

PCR with orf1 primers

rmp

Wet swabs

Dry swabs

33

96

(n=133)

Conclusion365
366
367
368
369
Positive 370
371
Negative 372
373
Positive 374
375
376
Negative
377
378
Positive 379
380

381
382

Table 3: Comparison of in-house PCR using dry swabs with wet swabs as mode of sample

383

collection, for N. gonorrhoeae