Manual

FTD Respiratory pathogens 33

32 reactions (catalog no. FTD-2P.3-32)

64 reactions (catalog no. FTD-2P.3-64)

Qualitative assay for in vitro diagnostics

For use with the ABI 7500, ABI 7500 Fast, ViiA7,
Bio-Rad CFX96, LightCycler®480, RotorGene 3000/6000/Q
and SmartCycler®

0123

FTD-2P.3-32, FTD-2P.3-64

Fast Track Diagnostics Luxembourg S.à.r.l.; 29, rue Henri Koch; L-4354
Esch-sur-Alzette; Luxembourg

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 FTD Respiratory pathogens 33

Table of Contents

1. IDENTIFICATION OF THE MANUFACTURER ............................................... 3

2. IDENTIFICATION OF THE PRODUCT .......................................................... 3
3. INTENDED USE ........................................................................................... 4
4. PATHOGEN INFORMATION ........................................................................ 5
5. CONTENTS ................................................................................................ 11
6. PRECAUTIONS AND WARNINGS .............................................................. 12
6.1 SAFETY INFORMATION ................................................................................ 12
6.2 HANDLING REQUIREMENTS .......................................................................... 12
6.3 SAFE WASTE DISPOSAL ............................................................................... 12
7. STORAGE AND STABILITY CONDITIONS.................................................. 13
8. PRINCIPLE OF THE METHOD .................................................................... 13
9. ADDITIONALLY REQUIRED EQUIPMENT .................................................. 14
10. SAMPLES .................................................................................................. 14
11. PROCEDURE ............................................................................................. 15
11.1 PRELIMINARY EXTRACTION PROCEDURE USING THE EASYMAG® ........................... 15
11.2 MAIN PCR SETUP PROCEDURE .................................................................... 16
12. PROGRAMMING OF THE THERMOCYCLER ................................................ 20
13. ASSAY VALIDATION ................................................................................. 23
14. SETUP ON THE ABI® 7500 ........................................................................ 24
15. INTERPRETATION OF RESULTS................................................................ 27
16. TROUBLESHOOTING ................................................................................ 29
17. VALIDATION ............................................................................................ 30
18. LEGEND OF SYMBOLS............................................................................... 30

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 FTD Respiratory pathogens 33

1. Identification of the manufacturer

Fast Track Diagnostics Luxembourg S.à.r.l.
29, rue Henri Koch
L-4354 Esch-sur-Alzette
Tel.: +352 281098-1
Fax: +352 281098-214
info@ftd-ltd.com

2. Identification of the product

FTD Respiratory pathogens 33

Category: Multiplex Real-Time PCR for detection of influenza A, influenza B,

influenza C; influenza A (H1N1)swl, parainfluenza viruses 1, 2, 3 and
4; coronaviruses NL63, 229E, OC43 and HKU1; human
metapneumoviruses A and B; rhinovirus; respiratory syncytial viruses
A and B ; adenovirus; enterovirus; parechovirus; bocavirus;
Pneumocystis jirovecii; Mycoplasma pneumoniae; Chlamydia
pneumoniae; Streptococcus pneumoniae; Haemophilus influenzae
type B; Staphylococcus aureus; Moraxella catarrhalis; Bordetella
spp.*; Klebsiella pneumoniae; Legionella pneumophila/Legionella
longbeachae; Salmonella species and Haemophilus influenzae
including internal control. *except Bordetella parapertussis

Reference: FTD-2P.3-32 Test for 32 reactions.

FTD-2P.3-64 Test for 64 reactions.

Reagents in the kits are sufficient for 32 or 64 reactions. These kit sizes allow
maximal flexibility from 1 to 30 patients in FTD-2P.3-32 and from 1 to 62
patients in FTD-2P.3-64. According PCR run amounts are shown in Table 1.

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 FTD Respiratory pathogens 33

Table 1: Minimum and maximum patient amounts and according run amounts possible for
FTD-2P.3-32 and FTD-2P.3-64.

FTD-2P.3-32 minimum maximum

amount patients 1 30
amount runs 10 1
FTD2P.2-64 minimum maximum
amount patients 1 62
amount runs 20 1

Indication: For in vitro diagnostics.

3. Intended use
FTD Respiratory pathogens 33 is an in vitro test with eight multiplex RT-PCR
reactions for the qualitative detection of the following viruses, bacteria and fungi
causing respiratory infections: influenza A, influenza B, influenza C; influenza A
(H1N1)swl, parainfluenza viruses 1, 2, 3 and 4; coronaviruses NL63, 229E, OC43
and HKU1; human metapneumoviruses A and B; rhinovirus; respiratory syncytial
viruses A and B; adenovirus; enterovirus; parechovirus; bocavirus; Pneumocystis
jirovecii; Mycoplasma pneumoniae; Chlamydia pneumoniae; Streptococcus
pneumoniae; Haemophilus influenzae type B; Staphylococcus aureus; Moraxella
catarrhalis; Bordetella spp.*; Klebsiella pneumoniae; Legionella pneumophila/
Legionella longbeachae; Salmonella species and Haemophilus influenzae.
*except Bordetella parapertussis

Be aware that BLAST results show that primers and probe targeting Legionella
pneumophila might also target Legionella worsleiensis and Legionella
fairfieldensis.

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4. Pathogen information
Influenza viruses belong to the family Orthomyxoviridae and resemble
encapsulated negative-sense single stranded RNA viruses. Influenza A (FLUA)
virus infection is associated with acute respiratory infections of varying severity,
ranging from asymptomatic infection to fatal disease. Typical influenza symptoms
include fever, sore throat, cough, headache and myalgia. Complications of
influenza infections include primary influenza viral pneumonitis, bacterial
pneumonia and exacerbation of underlying chronic conditions. Illness tends to be
most severe in the elderly, in infants and young children, and in
immunocompromised hosts. The swine-lineage influenza A virus subtype
H1N1 (=A(H1N1)swl) was reported in spring 2009. On June 11, 2009, the
WHO declared an H1N1 pandemic, marking the first global pandemic since the
1968 Hong Kong Flu. Influenza B (FLUB) viruses cause the same spectrum of
disease as influenza A. However, influenza B viruses are not known to cause
pandemics. Influenza C (FLUC) viruses cause mild upper respiratory tract
illness. Lower respiratory tract complications are rare. There is no vaccine against
influenza C virus. Influenza viruses are spread from person to person primarily
through large-particle respiratory droplet transmission. The typical incubation
period for influenza is 1-4 days.

Human rhinoviruses (RV) are the most frequent viral infective agents in
humans and the predominant cause of the common cold. Rhinoviruses have
single-stranded positive sense RNA genomes and are widespread in all age
groups where they can cause upper and lower respiratory tract infections.
Increased testing has recently implicated these viruses in severe infections such
as asthma and COPD. Although infections occur year-round, the incidence is
highest in the fall and the spring. There are two modes of transmission: via
aerosols of respiratory droplets and from contaminated surfaces, including direct
person-to-person contact.

Human metapneumoviruses A and B (HMPVA and B) are negative single-

stranded RNA virus causing a wide range of respiratory diseases, ranging from
mild upper respiratory tract infections to severe bronchiolitis and pneumonia.
HMPV is considered ubiquitous worldwide. In temperate regions, HMPV circulates
mainly during the winter. Although HMPV infections have been diagnosed in all
age groups, the virus likely has its greatest effect in children. Transmission
occurs by contact with contaminated secretions, via droplets, aerosols, or
fomites. Hospital acquired infections with Human metapneumovirus have also
been reported.

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Human parainfluenza viruses (HPIV1-4) belong to the enveloped single-

stranded RNA viruses and have been associated with every type of upper and
lower respiratory tract illness, including common cold with fever
laryngotracheobronchitis, bronchiolitis, and pneumonia. Para 1 and Para 2 are
the pathogens most commonly associated with croup, and Para 3 is the
pathogen most commonly associated with bronchiolitis and pneumonia in infants
and young children. Types 1 and 2 tend to occur mostly in the winter months
whereas type 3 occurs mainly in the spring. Little is known about the
epidemiology of parainfluenza 4. Generally, it has been noted that the rate of
infection is relatively the same in age groups from young infants to adults.
Human parainfluenza usually spread from person to person through the air by
coughing and sneezing and close personal contact with an incubation period of
approximately 4 days.

Human coronaviruses (HCoV-NL63 or Cor63, HCoV-229E or Cor 229, HCoV-

OC43 or Cor43 and HCoV-HKU1 or HKU1) are associated with a range of
respiratory outcomes, including bronchiolitis and pneumonia. Coronaviruses are
enveloped viruses with a positive-sense RNA genome and with a nucleocapsid.
HCoV-NL63 is associated with croup in children, whereas it is suggested that the
virus probably causes the common cold in healthy adults. HCoV-229E is a proven
common cold virus in healthy adults, and it is likely that both viruses induce
comparable symptoms in adults, even though their modes of infection differ.
HCoV-OC43 is generally characterized by sore throats. HCoV-HKU1 causes mild
upper respiratory diseases, the common cold, bronchiolitis, and pneumonia, with
symptoms such as rhinorrhoea, fever, cough, febrile seizure, and wheezing.
Infection occurs through the air by coughing and sneezing, and close personal
contact, such as touching or shaking hands with an incubation period of
approximately 4 days.

Adenoviruses (HAdV) consist of non-enveloped dsDNA and are a common

cause of respiratory illness. The symptoms can range from the common cold to
pneumonia, croup, and bronchitis. Depending on the type, adenoviruses can
cause other illnesses such as gastroenteritis, conjunctivitis, cystitis, and less
commonly, neurological diseases. Adenoviral infections affect infants and young
children much more frequently than adults. Severe, disseminated infection can
occur in immunocompromised subjects. Adenoviruses are responsible for 15% of
children that are hospitalized with gastroenteritis.

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Respiratory syncytial viruses A and B (HRSVA and HRSVB) are an important

cause of lower-respiratory-tract infection in all age groups. The genome consists
of a single strand of RNA with negative polarity. Most infections occur during the
winter season (December-March in the Northern hemisphere). HRSVs are of
particular importance as a cause of severe lower respiratory tract infection in
infants (causing bronchiolitis and pneumonia), immunocompromised and the
elderly. HRSV can be spread when droplets containing the virus are sneezed or
coughed into the air. Infection can also result from direct and indirect contact
with nasal or oral secretions. People infected with HRSV are usually contagious
for 3 to 8 days; most otherwise healthy people recover from HRSV infections in 1
to 2 weeks. However, infection can be severe in infants, young children, and
older adults.

Enteroviruses (EV) are a genus of positive-sense single-stranded RNA viruses

including polioviruses, coxsackieviruses, echoviruses, and other enteroviruses.
Non-polio enteroviruses are very common. They are second only to the "common
cold" viruses, rhinoviruses, as the most common viral infectious agents in
humans. EV infections are most likely to occur during the summer and fall. EV
affect millions of people worldwide each year, and are often found in the
respiratory secretions (e.g., saliva, sputum, or nasal mucus) and stool of an
infected person. No vaccine is currently available for the non-polio enteroviruses.

Human bocavirus (HBoV) is a ssDNA virus belonging to the Parvoviridae family

and has been found in infants and children with respiratory tract illness in all
areas of the world. Symptoms of respiratory tract infection, gastrointestinal
symptoms and skin rash were reported. It is suggested that bocavirus is mainly
spread to other humans by respiratory secretion. However, it can also be found
in stools and in blood, which may be alternative ways of virus spreading.

Human parechoviruses (HPeV) are positive ssRNA viruses and are prevalent in
young children. They have been associated with respiratory disease, including
upper and lower respiratory tract disease. It has also been claimed that they
commonly cause mild gastroenteritis and, less frequently, meningitis and
neonatal sepsis.

Chlamydia pneumoniae (Cpneu) is emerging as a significant cause of

respiratory disease, including pneumonia and bronchitis in humans. It is a gram
negative aerobic, intracellular pathogen. The organism has also been implicated
as an infectious trigger for asthma. It is typically acquired by otherwise healthy
people and is a form of community-acquired pneumonia.

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Mycoplasma pneumoniae (Mpneu) is a common cause of upper respiratory

tract infections with fever, cough, malaise, and headache. The bacterium lacks a
peptidoglycan cell wall and can therefore not be detected by gram staining.
Radiologically confirmed pneumonia develops in 5-10 % of the cases; also rare
extrapulmonary syndromes, including cardiologic, neurologic, and dermatologic
findings are possible. The transmission is by person-to-person contact with
respiratory secretions. The incubation period is between 1 to 4 weeks.

Staphylococcus aureus (Saur), a spherical gram-positive bacterium, is

frequently found in the nose, in the throat and on the skin. Saur is a part of the
normal human flora. Approximately 30 % of the population are persistently
colonized. Saur is a leading cause of nosocomial infections. It is the most
common cause of surgical wound infections that can lead to bacteremia. In the
community, Saur remains an important cause of skin and soft tissue infections,
respiratory infections, and (among injection drug users) infective endocarditis.

Streptococcus pneumoniae (Spneu), a Gram-positive diplococcus, causes

diseases such as sepsis, meningitis, and pneumonia. The organism is one of the
most common bacteria seen in community-acquired pneumonias, accounting for
up to 25% of these infections. It is also one of the top two isolates found in ear
infection and otitis media. Pneumococcal pneumonia is more common in the very
young and the very old.

Klebsiella pneumoniae (Kpneu) is non-motile, gram-negative bacterium and

an emerging pathogen with serious clinical and infection control implications. It is
clinically the most significant member of the Klebsiella genus of
Enterobacteriaceae. The most common diseases are infections of the respiratory
or urinary tract. Klebsiellae have also been incriminated in nosocomial infections.
The most common infection caused by Kpneu outside the hospital is pneumonia,
typically in the form of bronchopneumonia and also bronchitis. These patients
have an increased tendency to develop lung abscess, cavitation, and empyema.

Moraxella catarrhalis (Morax), a gram-negative aerobic dipolococcus is an

important bacterial cause of otitis media in children and respiratory tract
infections in the elderly. Moreover, Morax is an important cause of lower
respiratory tract infections, particularly in adults with COPD. In
immunocompromised hosts, the bacterium can cause a variety of severe
infections including pneumonia, endocarditis, septicemia, and meningitis.

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Haemophilus influenzae (Haeinf) is a non-motile, Gram-negative, rod-shaped

bacterium. Haeinf is responsible for a wide range of clinical diseases.
Encapsulated strains were classified on the basis of their distinct capsular
antigens. There are six generally recognized types of encapsulated Haeinf: a-f.
Unencapsulated strains are termed nontypeable (NTHi) because they lack
capsular serotypes. They are almost always less invasive; they can, however,
produce an inflammatory response in humans, which can lead to many
symptoms. They can cause media, conjunctivitis, and sinusitis in children, and
are associated with pneumonia and meningitis. Most strains of Haeinf are
opportunistic pathogens. One clinical very important member of the encapsulated
strains is Haemophilus influenzae Type B (HIB) which causes bacteremia,
pneumonia and acute bacterial meningitis. On occasion, it causes cellulitis,
osteomyelitis, epiglottitis and infectious arthritis.

Legionella pneumophila (Lpneu) is a pathogenic Gram-negative bacterium

that causes Legionellosis or Legionnaires’ disease and Pontiac fever (flu-like
illness). L. pneumophila parasites and proliferates within free-living protozoa.
Common sources of infection are typically associated with water systems such as
cooling towers, spas, showers, and other warm water systems. Natural sources
of L. pneumophila include freshwater ponds and creeks. Risk factors for
infections are smoking, preexisting medical conditions, and immunosuppression.
The clinical manifestations of legionellosis range from no symptoms to acute
atypical pneumonia and multisystem disease. Advanced stages of the disease
cause problems with the gastrointestinal tract and the nervous system and lead
to diarrhea and nausea.

Legionella longbeachae (Llong) is a pathogenic Gram-negative bacterium that

causes Legionellosis or Legionnaires’ disease and Pontiac fever (flu-like illness).
The main sources of infection are soils, composts, and potting mixes. Risk factors
for infections are smoking, preexisting medical conditions, and
immunosuppression. The clinical manifestations of legionellosis range from no
symptoms to acute atypical pneumonia and multisystem disease. Advanced
stages of the disease cause problems with the gastrointestinal tract and the
nervous system and lead to diarrhea and nausea.

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Pneumocystis jiroveci (PCP), a yeast-like fungus of the genus Pneumocystis,

is one of several organisms known to cause life-threatening opportunistic
infections in immunocompromised patients worldwide. By contrast, PCP occurs
only very rarely in immunocompetent individuals. Symptoms include fever, non-
productive cough, shortness of breath and weight loss. In a small number of
cases, the fungus can invade other visceral organs, such as the liver, spleen and
kidney. Although animal studies suggest that the principal infection route is by
air, the exact mode of transmission remains unknown.

Bordetella pertussis (Bord) is a Gram-negative, aerobic coccobacillus of the

genus Bordetella, and the causative agent of pertussis or whooping cough. The
infection occurs mostly in children under the age of one when they are
unimmunized, or children with faded immunity, normally around the ages 11
through 18. The signs and symptoms are similar to a common cold: runny nose,
sneezing, mild cough, and low-grade fever.

Salmonella species (Salm) are Gram negative, non-lactose fermenting and non-
sporing bacteria. They are aetiologic agents of the typhoid fever and paratyphoid
fever, and the food-borne disease salmonellosis. Common sources of infection
include poultry meat and meat products, eggs and egg products. Some of the
symptoms of salmonellosis are diarrhoea, vomiting, fever, and abdominal pain.
They are also known to cause pneumonia.

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5. Contents
Table 2: Table of contents: PP = primer and probe, IC = internal control, PC = positive
control, NC = negative control.

Contents FTD-2P.3-32 FTD-2P.3-64

FluRhino PP Primer/probe mix for FLUA, FLUB, H1N1 & RV 1 x 48 µl 2 x 48 µl

COR PP Primer/probe mix for Cor43, 63, 229 & HKU1 1 x 48 µl 2 x 48 µl

Primer/probe mix for HPIV2-4 & IC (EAV)
ParaEAV PP 1 x 48 µl 2 x 48 µl
Primer/probe mix for HBoV, Mpneu, HPIV1 &
BoMpPf1 PP 1 x 48 µl 2 x 48 µl
HMPVA/B
Primer/probe mix for HRSVA & B, HAdV, EV &
RsEPA PP 1 x 48 µl 2 x 48 µl
HPeV
Primer/probe mix for Saur, Spneu, Cpneu & HIB
RespBac PP 1 x 48 µl 2 x 48 µl
Primer/probe mix for Kpneu, Lpneu/Llong, PCP
KLePSa PP & Salm 1 x 48 µl 2 x 48 µl

Primer/probe mix for Morax, Bord, FLUC &

MoBoCH PP Haeinf 1 x 48 µl 2 x 48 µl

Positive control: plasmid pool for FLUA, FLUB,

H1N1swl, RV, Cor63, Cor229, Cor43, HKU,
Resp21 PC 1 x 750 µl 2 x 750 µl
HPIV1-4, HMPV, HBoV, Mpneu, HRSV, HAdV, EV
& HPeV
Positive control: plasmid pool for use with
PPmixes 6-8. This pool includes Saur, Spneu,
Resp33 PC2 1 x 450 µl 2 x 450 µl
Cpneu, HIB, Kpneu, Lpneu/Llong, PCP, Salm,
Morax, Bord, FLUC and Haeinf.
NC Negative control 1 x 4000 µl 1 x 8000 µl

IC Internal control 1 x 128 µl 2 x 128 µl

Enzyme 25x RT-PCR Enzyme mix (Fast-track mastermix) 1 x 256 µl 2 x 256 µl

Buffer 2x RT-PCR Buffer (Fast-track mastermix) 2 x 1600 µl 4 x 1600 µl

Each vial contains additional volume for pipetting inaccuracy. The box itself, the
cover of the box and each vial are labeled with a lot number.

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6. Precautions and warnings

6.1 Safety information

Warning notice: the negative control contains lysis buffer.

Hazardous pictogram:

Signal word: Warning

Hazardous statements:

H315: Causes skin irritation.

H317: May cause an allergic skin reaction.
H319: Causes serious eye irritation.

Precautionary statements:

Prevention:
P280: Wear protective gloves/protective clothing/eye protection/face protection.

6.2 Handling requirements

Use of this product should be limited to personnel trained in the techniques of
PCR. This product should be used in accordance with Good Laboratory Practice.
Take the normal precautions required for handling all laboratory reagents. Do not
mix reagents from different lots. Do not use the product after its expiration date.

6.3 Safe waste disposal

Dispose of unused reagents and waste in accordance with country, state or local
regulations.

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 FTD Respiratory pathogens 33

7. Storage and stability conditions

The components of the FTD product should be stored in the original packaging at
–20°C and are stable until the expiration date stated on the label. The product is
shipped in frozen packages which should ensure a transport temperature under
+10°C (satisfactory, according to stability studies). The reagents within a kit are
suitable for 32 or 64 reactions. Freeze the product immediately after usage. More
than 9x thawing and freezing of the reagents per tube should be avoided, as this
may reduce assay sensitivity. We recommend aliquoting the reagents according
to your needs after the first thawing.

For stability performance data, please refer to www.fast-trackdiagnostics.com.

8. Principle of the method

The viral RNA is transcribed into cDNA using a specific primer mediated reverse
transcription step followed immediately in the same tube by polymerase chain
reaction. The DNA of different pathogens is amplified simultaneously in the same
tube by polymerase chain reaction. The presence of specific pathogen sequences
in the reaction is detected by an increase in fluorescence observed from the
relevant dual-labeled probe, and is reported as a cycle threshold value (Ct) by
the Real-Time thermocycler. The assay uses Equine arteritis virus (EAV) as an
internal control (IC), which is introduced into each sample and the negative
control at the lysis buffer stage of the extraction process.

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9. Additionally required equipment

FTD kits are suited for use with the Applied Biosystems® 7500/7500Fast (Thermo
Fisher Scientific), CFX96™ (BIO-RAD), LightCycler®480 (Roche) and Rotor-Gene
3000, 6000, Q (Qiagen) and SmartCycler® (Cepheid; in combination with Life
Science software 2.0d). The assay has been fully validated on an Applied
Biosystems® 7500 with Fast-track mastermix and with the NucliSENS® easyMag®
(bioMérieux). If you want to use different extraction methods, please firstly
check their compatibility with FTD.

 For using the SmartCycler® we recommend the FTD smartmix.

 Disposable powder-free gloves
 Pipettes (adjustable)
 Sterile pipette tips with filters
 Vortex mixer
 Desktop centrifuge
 For the ABI® 7500, CFX96™ and LightCycler®480, 96 well PCR plates and
plate sealers are recommended. For the usage of the Rotor-Gene
3000/6000/ Q and SmartCycler® use appropriate tubes and caps.
 Sample rack

The validation file of Fast-track mastermix and a detailed compatibility list are
available under www.fast-trackdiagnostics.com.

10. Samples
This test is for use with extracted RNA and DNA from respiratory samples
(throat/nasal/nasopharyngeal swabs, bronchoalveolar lavage and sputum) of
human origin. In some cases other sample types may be tested including post
mortem material such as lung tissue. For long term storage FTD recommends to
store all samples at -20°C until extraction.

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11. Procedure

11.1 Preliminary extraction procedure using the easyMAG®

If you want to use different extraction methods, please firstly check their
compatibility at www.fast-trackdiagnostics.com.

Extraction of specimens and negative control with the easyMAG®:

1. Thaw the negative control (NC, white cap) and the internal control (IC,
dark blue cap). Before use, the reagents have to be thawed completely,
mixed (by short vortexing) and spun down briefly.

2. Extract your samples and the NC. We recommend a starting volume for
the extraction of 400 µl and an elution volume of 110 µl.

It is well recognised that sensitivity is increased if a larger volume of

clinical material is extracted into a small volume of eluate. This is
particularly important with samples where pathogen load is expected to be
low, such as CSF (and others). In this circumstance, we recommend an
input volume of at least 500 µl. Where extreme sensitivity is required,
such as certain clinical situations involving HIV or hepatitis viruses in
blood, up to 1ml should be extracted. Please follow manufacturer`s
extraction kit recommendations.

3. Add 4 µl internal control (IC, blue cap) directly to the lysis buffer of each
extraction. Never add the internal control directly to the sample unless
they are in lysis buffer. Adding the internal control to each of the samples
and to the negative control is a very important step to see if the nucleic
acid isolation has been successful and to check for possible PCR inhibition.

4. Do not extract positive controls as they are plasmids and will be inhibited.

5. Make sure to refreeze the left over volumes of NC and IC right after
usage.

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11.2 Main PCR setup procedure

Preparation of PCR with Fast-track mastermix:

1. Thaw reagents for the reaction: FluRhino PP, COR PP, ParaEAV PP,
BoMpPf1 PP, RsEPA PP, RespBac PP, KLePSa PP, MoBoCH PP, the positive
controls (PC) and 2x RT-PCR buffer (Fast-track mastermix, light blue cap)
of Fast-track mastermix. The PC and the extracted NC have to be included
in each run. Before use, the reagents have to be thawed completely,
mixed (by short vortexing) and spun down briefly. The positive controls
need to be thawed at room temperature for 20-30 minutes and vortexed
thoroughly right before use. Make sure to keep 25x RT-PCR enzyme (Fast-
track mastermix, orange cap) of Fast-track mastermix in a freezer or on a
cooling block at all times.

2. Pipette the required amount of 2x RT-PCR buffer in a 1.5ml tube. Do not

immerge the whole tip into the liquid when pipetting 2x RT-PCR buffer to
avoid waste of material and to obtain accurate volumes. Pipetting must be
done very slowly to prevent air bubbles. Wipe the tip against the edge of
the vessel to remove excess liquid outside the tip before dispensing.

3. Add according amount (see Table 3) of FluRhino PP, COR PP, ParaEAV PP,
BoMpPf1 PP, RsEPA PP, RespBac PP, KLePSa PP and MoBoCH PP to 2x RT-
PCR buffer. Take care to change the tips after each pipetting step.

4. Pipette the required amount (see Table 3) of 25x RT-PCR enzyme to

FluRhino PP, COR PP, ParaEAV PP, BoMpPf1 PP, RsEPA PP, RespBac PP,
KLePSa PP and MoBoCH PP with 2x RT-PCR buffer (reaction mix). Do not
immerge the whole tip into the liquid when pipetting 25x RT-PCR enzyme
to avoid waste of material and to obtain accurate volumes. Pipetting must
be done very slowly to prevent air bubbles. Wipe the tip against the edge
of the vessel to remove excess liquid outside the tip before dispensing.
Take care to change the tips after each pipetting step. Vortex the
complete master mix briefly and spin it down. If you use the SmartCycler®
please add per reaction 4µl of FTD smartmix to reaction mix.

5. Make sure to refreeze the remaining volumes of PP, PC and 2x RT-PCR

buffer (Fast-track mastermix) after usage.

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Table 3: Shown are the amounts of reagents that are needed for 1, 15, 32 and 64 wells.

FTD-2P.3-32: Each PPmix is sufficient for 32 reactions (+ pipetting inaccuracy). A minimum of

1 patient up to maximum 30 patients plus PC and NC is possible.
FTD-2P.3-64: Each PPmix is sufficient for 64 reactions (+ pipetting inaccuracy). A minimum of
1 patient up to maximum 62 patients plus PC and NC is possible.

Number of reactions 1 15 32 64
Buffer 12.5 µl 187.5 µl 400 µl 800 µl
FTD-2P.3- PPmix 1.5 µl 22.5 µl 48 µl 96 µl
32/64 Enzyme 1 µl 15 µl 32 µl 64 µl
Total 15 µl 225 µl 480 µl 960 µl

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 FTD Respiratory pathogens 33

Preparation of a 96 well plate for the ABI® 7500:

All our tests are validated on ABI® 7500, Bio-Rad CFX96™, LightCycler®480 and
RotorGene. If you intend to use the Bio-Rad CFX96™ or the LightCycler®480 you
must use appropriate plates and adhesive films. For RotorGene and SmartCycler®
use adequate tubes and caps. If you intend to run our tests on a different cycler,
please firstly refer to: www.fast-trackdiagnostics.com.

Preparation of a 96 well plate for ABI® 7500

1. Take a 96 well plate which is compatible with the ABI® 7500.
2. Pipette 15 µl of the reaction mix (FluRhino PP) in the wells.
3. Pipette 15 µl of the reaction mix (COR PP) in the wells.
4. Pipette 15 µl of the reaction mix (ParaEAV PP) in the wells.
5. Pipette 15 µl of the reaction mix (BoMpPf1 PP) in the wells.
6. Pipette 15 µl of the reaction mix (RsEPA PP) in the wells.
7. Pipette 15 µl of the reaction mix (RespBac PP) in the wells.
8. Pipette 15 µl of the reaction mix (KLePSa PP) in the wells.
9. Pipette 15 µl of the reaction mix (MoBoCH PP) in the wells.
10. Add 10 µl of the extracted samples, the extracted negative control and the
positive control (which is not extracted; thaw at room temperature for 20-
30 minutes and vortex thoroughly right before use). Each run must include
a negative and a positive control.
11. Mix briefly by pipetting up and down.
12. Close the plate with the ABI optical adhesive film.
13. Slightly vortex the plate and centrifuge briefly afterward.
14. Put the plate in the ABI®.7500.
15. Figure 1 (10 patients + PCs + NC) shows an example for location of
samples and controls on an ABI 7500 plate.

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 FTD Respiratory pathogens 33

Figure 1: Schematic presentation of an example for location of samples and controls

on a 96 well plate for the ABI® 7500.
Rows A-H; columns 1-12= layout of the 96 well plate
S1; S2; S3; S4,…, S12= mastermix and samples 1-12
PC= Master mix and positive control
NC= Master mix and negative control
Reaction mix with: FluRhino PP (row A)
COR PP (row B)
ParaEAV PP (row C)
BoMpPf1 PP (row D)
RsEPA PP (row E)
RespBac PP (row F)
KLePSa PP (row G)
MoBoCH PP (row H)

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 FTD Respiratory pathogens 33

12. Programming of the thermocycler

Pay particular attention to the settings for the detectors:

Table 4: Settings of the detectors.

Detection wavelength
PP mix Pathogen Dye
(nm)*
FLUA green 520
RV yellow 550
FluRhino
FLUB orange 610
H1N1 red 670
Cor 229 green 520
Cor 63 yellow 550
COR
HKU1 orange 610
Cor 43 red 670
HPIV3 green 520
HPIV2 yellow 550
ParaEAV
HPIV4 orange 610
IC (EAV) red 670
HPIV1 green 520
HMPV A/B yellow 550
BoMpPf1
HBoV orange 610
Mpneu red 670
HRSVA/B green 520
HPeV yellow 550
RsEPA
EV orange 610
HAdV red 670
S.aur green 520
C.pneu yellow 550
RespBac
HiB orange 610
S.pneu red 670
PCP green 520
Lpneu/Llong yellow 550
KLePSa
K.pneu orange 610
Salm red 670
Morax green 520
FLUC yellow 550
MoBoCH
Bord orange 610
Haeinf red 670

*The mentioned detection wavelengths are from the ABI® 7500. They can be
slightly different on other machines.

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 FTD Respiratory pathogens 33

NEW! Fast-track mastermix PCR programme 2:

50°C for 15 minutes hold

94°C for 1 minute hold
40 cycles of: 94°C for 8 second
60°C for 1 minute

FTDliquid kits and FTlyo kits usage:

 To use FTlyo and FTDliquid kits on one plate or FTlyo kits alone, use the
PCR programme 2.
 To use only FTDliquid kits, FTD is recommending the optimized PCR
programme 2, as all validations are done with this program. But be aware,
that you still can use the former PCR programme 1 as well.

Fast-track mastermix PCR programme 1:

42°C for 15 minutes hold

94°C for 3 minutes hold

40 cycles of: 94°C for 8 seconds

60°C for 34 seconds

Detailed information on programming of the thermocyclers is provided in the

instruction manuals of the cyclers which can be downloaded from our homepage
www.fast-trackdiagnostics.com.

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IMPORTANT NOTES:

If you use the ABI® 7500, it is necessary to change the setting for the passive
reference dye. (By default, the ROX dye is selected). After the step specifying the
detectors and task for each well, click finish and the software will create the
plate document. Click on a well, or click-drag, to select replicate wells. Enter the
sample name and change the passive reference to “none”.

If you use the ABI®7500 Fast, do NOT use the fast programme.

If you use the RotorGene turn off auto-gain optimization and set gains for
yellow, orange, red and green channels on 5.

If you use the LightCycler®480, it is necessary for you to perform one FTD
color compensation run before you start using FTD tests. FTD advises to run
a new FTD color compensation annually and after each maintenance of your
device. The reagents for FTD color compensation are supplied by FTD. Use Abs
Quant/Fit Points for analysis of the run. FTD recommends the usage of
transparent 96well plates.

If you use the SmartCycler®, please be aware that it is currently validated in

combination with the Cepheid, Life Science software 2.0d only and with the
FTD smartmix (FTD). If you want to use different enzymes please refer to FTD.

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 FTD Respiratory pathogens 33

13. Assay validation

Set a threshold as follows:

1. All negative controls should be below the threshold. If there is a potential

contamination (appearance of a curve in the negative control or a cluster of
curves in specimens at high Ct – for example above 36), results obtained are not
interpretable and the whole run (including extraction) has to be repeated.

2. All the positive controls must show a positive (i.e. exponential) amplification
trace. The positive controls must fall below a Ct of 33 (detailed information see
Interpretation of results).

3. Check the “component” trace before accepting the exponential trace as real.
Contact the equipment manufacturer or FTD for advice (support@fast-
trackdiagnostics.com).

4. All internal controls must show a positive (i.e. exponential) amplification

trace. The internal control must fall below a Ct of 33. If the internal control is
above CT 33, this points to a purification problem or a strong positive sample
that can inhibit the IC.

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 FTD Respiratory pathogens 33

14. Setup on the ABI® 7500

1. Open your experiment

2. On the drop down menu on the left choose “Analysis”

[A] and “Amplification Plot” [B].

3. Modify the “Graph Type” [C] as you prefer to “Linear” or

“Log” and the “Color” [D] to “Target”.

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 FTD Respiratory pathogens 33

4. In the top right corner of your screen choose “Analysis settings” [E].

5. A new window opens: Analysis settings; highlight all targets of all tests [F].

6. Unclick “Use Default Settings”, “Automatic Threshold” and “Automatic

Baseline” [G] and “Apply Analysis Settings” [H].

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 FTD Respiratory pathogens 33

7. For advanced analysis, you can also change your settings for each target in
the options window. Here you can modify the threshold and baseline for every
single parameter [I].

8. Check the positive controls, negative controls and internal controls first. They
have to follow the specifications mentioned in point 13 (Assay validation).

9. If all controls meet the specified ranges, check your samples for positive
traces.

10. Your Ct results for all color channels will be displayed on the “View Well
Table” window.

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 FTD Respiratory pathogens 33

15. Interpretation of results

The positive controls and any positive samples will show an exponential
fluorescence trace. Any specimen displaying an exponential trace is considered
as positive. For example, if a sample shows an exponential fluorescence trace at
a wavelength of ~550 (yellow channel) with KLePSa PP it contains Legionella
pneumophila and/or Legionella longbeachae DNA. BLAST results show that
primers and probe targeting Legionella pneumophila might also target Legionella
worsleiensis and Legionella fairfieldensis.

See Table 5 for detailed information of each pathogen of FTD Respiratory

pathogens 33. Pay attention to the exceptional cases mentioned in the table!

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 FTD Respiratory pathogens 33

Table 5: Possible results with FTD Respiratory pathogens 33.

Pos= positive; empty= negative

signal in signal in signal in signal in

PP mix Pathogen green yellow orange red
channel channel channel channel
FLUA POS
RV POS
FLUB POS
FluRhino PP H1N1 POS
If FluA AND H1N1 are positive, the patient is H1N1 positive. If just FluA is positive the patient is
FluA positive. If just H1N1 is positive the patient is H1N1 positive.
If Rhino AND EV (RsEPA PP) are positive the patient is EV positive. If just Rhino is positive the
patient is Rhino positive.
Cor229 POS
Cor63 POS
COR PP
HKU1 POS
Cor43 POS
HPIV3 POS
HPIV2 POS
ParaEAV PP HPIV4 POS
IC (EAV) POS
The IC has to be positive for each extracted material (patients and NC).
HPIV1 POS
HMPV A/B POS
BoMpPf1 PP
HBoV POS
Mpneu POS
RSVA&B POS
HPeV POS
RsEPA PP EV POS
HAdV POS
If just EV (no Rhino; FluRhino PP) is positive the patient is EV positive. If Rhino (FluRhino PP) AND
EV are positive the patient is EV positive.
Saur POS
Cpneu POS
RespBac PP
HIB POS
Spneu POS
PCP POS
Lpneu/Llong POS
Kpneu POS
KLePSa PP Salm POS
The Kpneu assay also detects K. variicola (98%) which is rarely found in clinical isolates and not
important as a human pathogen. Be aware that BLAST results show that primers and probe
targeting Legionella pneumophila might also target Legionella worsleiensis and Legionella
fairfieldensis.
Morax POS
FLUC POS
MoBoCH PP Bord POS
Haeinf POS
If you have a positive Haeinf result and a positive HIB (RespBac PP) result the patient is positive
for Haemophilus influenzae type B.

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 FTD Respiratory pathogens 33
16. Troubleshooting
No signal with positive controls
 Incorrect programming of the temperature profile of the thermocycler
 Compare the temperature profile to the manual.
 Incorrect configuration of the PCR reaction
 Check your work steps by means of the pipetting scheme and
repeat the PCR if necessary.
 Check calibration of pipettes.
 Incorrect handling of the positive controls
 Inadequate or no vortexing and thawing at room temperature
 The storage conditions for one or more product components did not
comply with the instructions or the FTD kit has expired.
 Please, check the storage conditions and the expiration date (see
the product label) of the reagents and use a new test, if necessary.

Weak or no signal of the internal control

 The PCR conditions do not comply with the protocol.
 Check the PCR conditions and repeat the PCR with correct settings
if necessary.
 The PCR was inhibited or no / too little internal control was added during
the extraction.
 Make sure that your extraction method is compatible with FTD kits.
 A strong positive signal of a pathogen can occasionally inhibit the
fluorescence of an internal control.

Signals within the negative control

 A contamination occurred during preparation of the PCR or during
extraction
 Repeat the PCR with new reagents in replicates.
 We recommend to pipette the positive controls last.
 Make sure that work space and instruments are decontaminated at
regular intervals.

If you have any further questions or if you encounter problems, please contact
support@fast-trackdiagnostics.com.

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17. Validation
For detailed validation data such as sensitivity, specificity, clinical studies and
external quality panel results, please refer to the related validation file at
www.fast-trackdiagnostics.com.

18. Legend of symbols

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