FTD Respiratory pathogens 21 - Mikrogen

Manual 

24 (catalog no. FTD 2-24/4) 



Qualitative assay for in vitro diagnostics 

FTD Respiratory pathogens 21 version 3/ March 2012 english 

FTD Respiratory pathogens 21 

For use with the 

ABI 7500, Bio-Rad CFX96, LightCycler ® 480, 

RotorGene 6000/ Rotor-Gene Q 

FTD 2-24/4; FTD 2-48/6; FTD 2-96/12 

Fast-track Diagnostics; 38, rue Hiehl; L-6131 Junglinster; Luxembourg 

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Table of Contents 



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

2. IDENTIFICATION OF THE PRODUCT .......................................................... 3 

3. INTENDED USE ........................................................................................... 3 

4. PATHOGEN INFORMATION ........................................................................ 4 

5. CONTENTS .................................................................................................. 6 

6. PRECAUTIONS AND WARNINGS ................................................................ 7 

6.1 SAFETY INFORMATIONS ................................................................................ 7 

6.2 HANDLING REQUIREMENTS ............................................................................ 7 

6.3 SAFE WASTE DISPOSAL ................................................................................. 7 

7. STORAGE AND STABILITY CONDITIONS .................................................... 7 

8. PRINCIPLE OF THE METHOD ...................................................................... 8 

9. ADDITIONALLY REQUIRED EQUIPMENT .................................................... 8 

10. SAMPLES .................................................................................................... 9 

11. PROCEDURE ............................................................................................... 9 

11.1 PRELIMINARY PROCEDURE WITH USING THE EASYMAG ....................................... 9 

11.2 MAIN PCR SETUP PROCEDURE WITH USING THE AGPATH ONE-STEP RT-PCR KIT ... 10 

12. PROGRAMMING OF THE THERMO CYCLER ............................................... 14 

13. ASSAY VALIDATION ................................................................................. 15 

14. CALCULATION OF THE RESULTS ON THE ABI 7500 .................................. 16 

15. INTERPRETATION OF RESULTS ................................................................ 18 

16. TROUBLESHOOTING ................................................................................ 20 

17. VALIDATION ............................................................................................ 21 

17.1. SPECIFICITY .................................................................................... 21 

17.2. SENSITIVITY .................................................................................... 22 

17.3. CLINICAL EVALUATION ........................................................................ 24 

18. LEGEND OF SYMBOLS ............................................................................... 26 

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1. Identification of the manufacturer 

Fast-track Diagnostics Luxembourg S.à.r.l. 

38, rue Hiehl 

Z. I. Laangwiss 

L-6131 Junglinster 

Tel. +352 780290 329 

Fax: +352 780290 514 

info@fast-trackdiagnostics.com 

2. Identification of the product 




Category: Multiplex PCR for detection of respiratory pathogens – 

including internal control 

Reference: FTD 2-24/4 Test for 24 patients, 6 assays, each for 4 specimens 

FTD 2-48/6 Test for 48 patients, 8 assays, each for 6 specimens 

FTD 2-96/12 Test for 96 patients, 8 assays, each for 12 specimens 

Indication: For in vitro diagnostics. 

3. Intended use 

The FTD Respiratory pathogens 21 (FTD Resp 21) is an in vitro test with five 

multiplex RT-PCR reactions for the detection of: influenza A (FluA), A(H1N1)swl 

(H1N1), B (FluB); coronaviruses NL63 (Cor63), 229E (Cor229), OC43 (Cor43) and 

HKU1 (HKU), parainfluenza 1, 2, 3 and 4 (Para1, Para2, Para3, Para4), human 

metapneumovirus A and B (HMPVA and B), rhinovirus (Rhino), respiratory 

syncytial viruses A and B (RSVA and B), adenovirus (AV), enterovirus (EV), 

parechovirus (PV), bocavirus (HboV), Mycoplasma pneumoniae (Mpneu) as an 

aid in the evaluation of infections by respiratory pathogens. 

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4. Pathogen information 



FluA and FluB are important viral infections of the respiratory tract of adults and 

children. Most cases occur during the annual winter epidemics. Severe infections 

can occur in the immunocompromised, those with chronic cardiac, pulmonary or 

metabolic disease and in the extremes of age. Most symptoms include fever, 

myalgia, sore throat and cough. In children gastrointestinal symptoms can also 

be present. Complications of influenza infection include primary (viral) or 

secondary (bacterial) pneumonia, cardiac involvement and neurological illness 

(including encephalopathy, encephalitis and Reyes syndrome. The swine-lineage 

influenza A virus subtype H1N1 (=A(H1N1)swl) was reported in spring 

2009. 

Para1, 2, 3, and 4 are common causes of upper and lower respiratory tract 

infections. Types 1 and 2 tend to occur mostly in the winter months whereas 

type 3 occurs mainly in the spring. Type 1 and 2 are commonly associated with 

croup in young children. Type 3 is second only to RSV as a cause of bronchiolitis 

and pneumonia in infants. Please note that these pathogens can also cause 

severe infections in immunocompromised patients, especially type 3. 

Rhino cause about one half of colds. There are over 100 types of rhinovirus 

known to cause respiratory infection in humans. They are common in all age 

groups and can cause upper and lower respiratory tract infections. They are a 

significant cause of bronchiolitis. Increased testing has implicated these viruses 

in severe infections, asthma and COPD. Severe infections are also likely to occur 

in immunocompromised. 

Cor229, Cor63, Cor43 and HKU are frequent causes of the common cold 

(second only to rhinoviruses). There have also been reports of pneumonia in the 

elderly, infants and immunocompromised. Increased testing will probably lead to 

detection of coronaviruses in severe upper and lower respiratory tract infections 

in other patient groups. 

RSVA and RSVB are common causes of respiratory illness in all age groups. 

Most infections tend to occur during the winter season (December-March in the 

Northern hemisphere). RSV is of particular importance as a cause of severe lower 

respiratory tract infection in infants (causing bronchiolitis and pneumonia), 

immunocompromised (especially BMT patients) and the elderly. 

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HMPV causes a spectrum of respiratory illness, ranging from mild upper 

respiratory tract infections to severe bronchiolitis and pneumonia. HMPV is 

associated with a rate of community acquired respiratory illness similar to that of 

the parainfluenza viruses and influenza virus but substantially less than that 

associated with RSV. HMPV may be responsible for a significant portion of the 

>150000 bronchiolitis hospitalizations in infants and children younger than 5 

years. HMPV has been recently identified in children and adults with acute 

respiratory tract infections (ARTI) in various parts of the world. 

AV are a common cause of respiratory infection (ranging from pharyngitis to 

severe pneumonia), eye infections (conjunctivitis and kerato-conjunctivitis) and 

gastrointestinal illness. Adenoviral infections affect infants and young children 

much more frequently than adults. Severe, disseminated infection can occur in 

immunocompromised. Rarely, adenoviruses can be a cause of haemorrhagic 

cystitis which mostly occurs in immunocompromised but has also been found in 

immunocompetent. 

HboV is a parvovirus that is one of the most common viruses in respiratory 

samples. The age group most frequently affected appears to be children between 

the ages of six months and two years. Symptoms of respiratory tract infection, 

gastrointestinal symptoms and skin rash were reported. It is one of only 2 known 

human parvovirus pathogens; the other is parvovirus B19. In general, 

parvoviruses are more important as veterinary pathogens. 

EV are implicated in a number of different clinical illnesses. EV are associated 

with a febrile illness sometimes with a rubelliform rash. They also cause aseptic 

meningitis. Other disease associations include herpangina (vesicles in the throat), 

hand foot and mouth disease, Bornholm disease (painful chest pain) and 

myocarditis. Enterovirus infection in neonates can be severe. Some enterovirus 

types are associated with acute haemorrhagic conjunctivitis. EV are unusual in 

winter. 

PV have as the other picornaviruses a single-stranded RNA genome of positive 

polarity. 60% of PV infections occurred in children under 1 year of age. 

The major peaks are during late summer and autumn, resembling the epidemic 

pattern of enteroviruses, and less frequently, during the winter and early spring. 

Diarrhea is the most common clinical manifestation, followed by respiratory 

symptoms. Although CNS manifestations are rather rare in this patient group, an 

association of PV infections with encephalitis and flaccid paralysis has also been 

described. 

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Mpneu is a small bacterium in the class Mollicutes. Mpneu is a common cause of 

upper respiratory tract infections with fever, cough, malaise, and headache. It 

may lead to tracheobronchitis with fever and nonproductive cough: radiologically 

confirmed pneumonia develops in 5-10 % of cases; rare extrapulmonary 

syndromes, including cardiologic, neurologic, and dermatologic findings. The 

transmission is by person-to-person contact with respiratory secretions. 

Incubation period is 1 to 4 weeks. 

5. Contents 

Table 1: PP = primer and probe, IC = internal control, PC = positive control, NC = negative 

control 

Label Contents 

FTD 

2-24/4 


2-48/6 


2-96/12 

FluRhino Primer/probe mix for Influenza 6 x 11 µl 8 x 14 µl 8 x 24 µl 

PP A, B, H1N1 & Rhino 

Cor Primer/probe mix for Cor63, 6 x 11 µl 8 x 14 µl 8 x 24 µl 

PP Cor43, Cor229 & HKU 

Para.EAV Primer/probe mix for Para2, 3, 6 x 11 µl 8 x 14 µl 8 x 24 µl 

PP 4 & IC 

BoMpPf1 Primer/probe mix for Para1, 6 x 11 µl 8 x 14 µl 8 x 24 µl 

PP HMPV A & B, HboV & Mpneu 

RsEPA Primer/probe mix for RSVA & B, 6 x 11 µl 8 x 14 µl 8 x 24 µl 

PP AV, EV & PV 

Positive control: plasmid pool 6 x 55 µl 8 x 55 µl 8 x 55 µl 

Resp 

PC 

for FluA, FluB, H1N1swl, Rhino, 

Cor63, Cor229, Cor43, HKU, 

Para1, 2, 3, 4, HMPV, HboV, 

Mpneu , RSV, Adeno, EV & PV 

Resp 

NC 

Negative control 6 x 220 µl 8 x 220 µl 8 x 220 µl 

Resp 

IC 

Internal control 

6 x 11 µl 8 x 16 µl 8 x 29 µl 

The box itself, the cover of the box and each vial are labelled with a lot number. 

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6. Precautions and warnings 

6.1 Safety informations 



Warning notice: the negative control contains lysis buffer. 

Hazard description: Xn Harmful 

Risk phrases: 

R 22 Harmful if swallowed. 

R 36/38 Irritating to eyes and skin. 

Safety phrases: 

S 13 Keep away from food, drink and animal feeding stuffs. 

S 26 In case of contact with eyes rinse immediately with plenty of water 

and seek medical advice. 

S 36 Wear suitable protective clothing. 

S 46 If swallowed seek medical advice immediately and show this 

container or label. 

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 (durability 1 year). 

6.3 Safe waste disposal 

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

regulations. 

7. Storage and stability conditions 

This product should be stored in its original packaging at –20°C until the 

expiration date printed on the label. The product is shipped below 10°C. For 

stability performance data, please contact FTD. Freeze immediately after one 

column of reagents has been taken out. Each column of reagents within a kit is 

suitable for one test run of 4, 6 or 12 patients. Do not refreeze columns that 

have been taken out of the kit and thawed. 

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8. Principle of the method 



RNA is transcribed into cDNA using a specific primer mediated reverse 

transcription step followed immediately in the same tube by polymerase chain 

reaction. Detection of products is via a dual labelled molecular probe for each 

virus and bacteria of the multiplex PCR. The presence of specific viral and 

bacterial sequences in the reaction is detected by an increase in the fluorescence 

observed from the relevant dual-labelled probe, and is reported as a cycle 

threshold value (Ct) by the real time thermo cycler. 

The assay uses Equine arteritis virus (EAV) as an internal control (IC), which is 

introduced into the lysis buffer at the extraction stage of each sample and the 

negative control. The internal control has to be extracted. 

9. Additionally required equipment 

The FTD Resp 21 is suited for use with the ABI 7500 (Applied Biosystems ® ), Bio- 

Rad CFX96 (BIO-RAD), LightCycler ® 480 (Roche) and RotorGene 6000 (Qiagen 

former Life Science Corbett)/ Rotor-Gene Q (Qiagen). The assay has been fully 

validated on an ABI 7500 with the AgPath-ID One-Step RT-PCR Kit (Ambion ® ) 

and with the EasyMAG (bioMérieux). If you want to use different cyclers, 

enzymes or extraction methods, please firstly check their compatibility with FTD. 

• Real time PCR master mix and enzyme. We recommend the AgPath-ID 

One-Step RT-PCR Kit (Ambion ® , cat# 4387391). 

• Disposable powder-free gloves 

• Pipettes (adjustable) 

• Sterile pipette tips with filters 

• Vortex mixer 

• Desktop centrifuge 

• For the ABI 7500, Bio-Rad CFX96 and LightCycler ® 480 96 well PCR plates 

and plate sealers are recommended. For the usage of the RotorGene 

6000/ Rotor-Gene Q it is recommended to use 0.1 ml strip tubes and 

caps. 

• Sample rack 

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10. Samples 



This test is for use with extracted RNA and DNA from respiratory samples 

(throat/nasal swabs, bronchoalveolare lavage and sputum) of human origin. In 

some cases other sample types may be tested including post mortem material 

such as heart and lung. 

For long term storage FTD recommends to store all samples at -20°C until 

extraction. 

11. Procedure 

11.1 Preliminary procedure with using the EasyMAG 

If you want to use different extraction methods, please firstly check their 

compatibility with FTD! 

Extraction of specimens and negative control with the EasyMAG: 

1. Thaw one negative control (Resp NC, 220 µl aliquot, white cap) and one 

internal control (Resp IC, blue cap). 

Before use, the reagents have to be thawed completely, mixed (by 

pipetting or short vortexing) and spun down briefly! 

2. Extract 4, 6 or 12 samples or multiples of 4, 6, 12 samples and the NC. 

We recommend a starting volume for the extraction of 200 µl and an 

elution volume of 55 µl. 

To increase the sensitivity of the test, you can use a higher starting 

volume for the extraction of your specimens according to your extraction 

method i.e. a starting volume of 400 µl and an elution volume of 55 µl. 

3. Add 2 µl internal control (IC, blue cap) directly to the lysis buffer of each 

extraction. 

Adding the internal control to each of your samples and to the negative 

control is a very important step to review the nucleic acid isolation has 

been successful and to check for possible PCR inhibition! 

4. Do not extract positive controls! 

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11.2 Main PCR setup Procedure with using the AgPath One-Step RT- 

PCR Kit 

If you want to use a different enzyme to the AgPath-ID One-Step RT-PCR Kit 

please firstly check with FTD! 

Preparation of PCR with AgPath-ID One-Step RT-PCR Kit: 

1. Thaw reagents for the reaction: FluRhino PPmix, Cor PPmix, Para.EAV 

PPmix, BoMpPf1 PPmix and RsEPA PPmix, the positive control (Resp PC) 

and the 2xRT PCR buffer of AgPath-ID One-Step RT-PCR Kit. The PC`s 

and the extracted NC have to be included in each run. 

Make sure to keep the enzyme of the AgPath-ID One-Step RT-PCR Kit 

cool. It should be kept in a freezer or on a cooling block at all times! 

2. Reagents in each column are sufficient for: 

FTD-2-24/4 4 patients, including controls 



3. Pipette the required amount (see table 2 below) of the “2xRT PCR buffer” 

(AgPath-ID One-Step RT-PCR Kit) to the FluRhino PPmix, Cor PPmix, 

Para.EAV PPmix, BoMpPf1 PPmix and RsEPA PPmix. 

Take care to change the tips after each pipetting step. 

4. Pipette the required amount (see table 2 below) of the “25x RT-PCR 

enzyme mix” (AgPath-ID One-Step RT-PCR Kit) to FluRhino PPmix, Cor 

PPmix, Para.EAV PPmix, BoMpPf1 PPmix and RsEPA PPmix with the “2xRT 

PCR buffer”. Take care to change the tips after each pipetting step. Vortex 

the complete master mixes briefly and spin down in a centrifuge. 

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Table 2: Shown are the amounts of reagents that are needed for one well and for multiples 

accordant to the kit size. 

FTD-2-24/4: The PPmix contained in one tube is sufficient for 7 reactions and includes 4 

patients, 1 NC, 1 PC, 10 % pipetting inaccuracy. 

FTD-2-48/6: The PPmix contained in one tube is sufficient for 9 reactions and includes 6 


FTD-2-96/12: The PPmix contained in one tube is sufficient for 15.5 reactions and includes 12 


FTD-2 

24/4 


48/6 


96/12 

Number of samples 1 7 

Preparation of the 

PCR assay 

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PP mix 1.5 µl 10.5 µl 

Enzyme 1 µl 7 µl 

Buffer 12.5 µl 87.5 µl 

Total 15 µl 105 µl 

Number of samples 1 9 


PCR assay 


Enzyme 1 µl 9 µl 

Buffer 12.5 µl 112.5 µl 

Total 15 µl 135 µl 

Number of samples 1 15.5 


PCR assay 


Enzyme 1 µl 15.5 µl 

Buffer 12.5 µl 194 µl 

Total 15 µl 232.8 µl

Preparation of a 96 well plate for the ABI 7500: 



All our tests are validated on the ABI 7500, so you need to take care when using 

different cyclers. If you want to use the Bio-Rad CFX96 or the LightCycler ® 480 

you must use appropriate plates and adhesive films. For the RotorGene please 

use adequate tubes for the correlating rotor. If you intend to run our tests on a 

different cycler to those mentioned please firstly check with FTD! 

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 FluRhino PPmix with the “2xRT PCR buffer” and the “25x RT- 

PCR enzyme mix” in the wells. 

3. Pipette 15 µl of the Cor PPmix with the “2xRT PCR buffer” and the “25x RT-PCR 

enzyme mix” in the wells. 

4. Pipette 15 µl of the Para.EAV PPmix with the “2xRT PCR buffer” and the “25x RT- 


5. Pipette 15 µl of the BoMpPf1 PPmix with the “2xRT PCR buffer” and the “25x RT- 


6. Pipette 15 µl of the RsEPA PPmix with the “2xRT PCR buffer” and the “25x RT-PCR 

enzyme mix” in the wells. 

7. Add 10 µl of the extracted samples, the extracted negative control and the positive 

control (which is not extracted). 

Each run must include a negative and the positive control! 

8. Mix briefly by pipetting up and down. 

9. Close the plate with the ABI optical adhesive film. 

10. Centrifuge briefly. 

11. Put the plate in the ABI 7500. 

12. Figure 1 shows an example for location of samples and controls on an ABI 7500 

plate. 

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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 = Master mix and samples 1-4 

Resp PC = Master mix and positive controls: rows A-E 

Resp NC = Master mix and negative control 

yellow background (row A) = Master mix with FluRhino PPmix 

red background (row B) = Master mix with Cor PPmix 

blue background (row C) = Master mix with Para.EAV PPmix 

purple background (row D) = Master mix with BoMpPf1 PPmix 

green background (row E) = Master mix with RsEPA PPmix 

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12. Programming of the thermo cycler 

Pay particular attention to the settings for the detectors: 



Table 3: Settings for the detectors. 

FluA FAM green 

Detection wave 

length (nm) * 

~ 520 

Rhino VIC yellow ~ 550 

FluB ROX orange ~ 610 

H1N1 Cy5 red ~ 670 

Cor 229 FAM green ~ 520 

Cor 63 VIC yellow ~ 550 

HKU ROX orange ~ 670 

Cor 43 Cy5 red ~ 610 

Para3 FAM green ~ 520 

Para2 VIC yellow ~ 550 

Para4 ROX orange ~ 670 

IC (EAV) Cy5 red ~ 610 

Para1 FAM green ~ 520 

HMPV A/B VIC yellow ~ 550 

HboV ROX orange ~ 670 

Mpneu Cy5 red ~ 610 

RSVA/B FAM green ~ 520 

PV VIC yellow ~ 550 

EV ROX orange ~ 670 

AV Cy5 red ~ 610 

PP mix Pathogen Reporter Dye 

FluRhino 

PP 

Cor 

PP 

Para.EAV 

PP 

BoMpPf1 

PP 

RsEPA 

PP 

* The mentioned detection wave lengths are from the ABI 7500. They can be 

slightly different on other machines. 

PCR programme: 

50°C for 15 minutes hold 

95°C for 10 minutes hold 

40 cycles of: 95°C, 8 sec. 

60°C, 34 sec. 

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The PCR programme might differ if using another enzyme than recommended. 

Detailed information on programming of the thermo cyclers is provided in the 

instruction manuals of the cyclers. 

NOTE: 

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”. 

NOTE: If you use the LightCycler ® 480, it is necessary for you to perform one 

color compensation run before you start using FTD tests. The reagents for 

the color compensation run have to be ordered from FTD. 

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), results obtained are not interpretable 

and the whole run (including extraction) has to be repeated. 

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

trace. The positive controls must fall below a Ct of 33. The stated spreading is 

based on the variance of the instrument. 

3. Check the “component” trace before accepting the exponential trace as real. 

Contact the equipment manufacturer or FTD for advice (technical@fasttrackdiagnostics.com). 

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

trace. The internal control must fall below a Ct of 33. The stated spreading is 

based on the variance of the instrument and the purification. If the internal 

control falls out of this range, this points to a purification problem. 

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14. Calculation of the results on the ABI 7500 

1. Open your experiment. 

2. In 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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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). 

7. 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). 

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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 clinical samples for 

positives. 

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

Table” window. 

15. Interpretation of results 

The positive control and any positive samples will show an exponential 

fluorescence trace. Any specimens displaying an exponential trace are considered 

as positive. For example, if a sample (e.g. with the FluRhino PPmix) shows an 

exponential fluorescence trace at a wavelength ~520 (FAM) it means that it 

contains FluA nucleic acid. 

If no FluA is detectable the sample can be considered as negative for FluA. 

See table 4 for detailed informations for each pathogen of the FTD Resp 21. 

Pay attention to the exceptional cases mentioned in the table! 

- 18 -



Table 4: Possible results with the FTD Resp 21. Pos = positive; empty = negative 

PP mix Pathogen Reporter 

FluRhino 

Cor 

Para.EAV 

BoMpPf1 

RsEPA 

signal in 

green 

channel 

FluA FAM POS 

- 19 - 

signal in 

yellow 

channel 

Rhino VIC POS 

signal in 

orange 

channel 

FluB ROX POS 

signal in 

red 

channel 

H1N1 Cy5 POS 

If Flu A AND FluA(H1N1)swl are positive, the patient is FluA(H1N1)positive. If just FluA 

is positive the patient is FluA positive. 

If Rhino AND EV (RsEPA PPmix) are positive the patient is EV positive. If just Rhino is 

positive the patient is Rhino positive. 

Cor 229 FAM POS 

Cor 63 YAK POS 

HKU ROX POS 

Cor 43 Cy5 POS 

Para3 FAM POS 

Para2 VIC POS 

Para4 ROX POS 

IC (EAV) Cy5 POS 

The IC has to be positive for each extracted material (patients and NC). 

Para1 FAM POS 

HMPV A&B VIC POS 

HboV ROX POS 

Mpneu Cy5 POS 

RSVA&B FAM POS 

PV VIC POS 

EV ROX POS 

AV Cy5 POS 

If just EV (no Rhino; FluRhino PPmix) is positive the patient is EV positive.

16. Troubleshooting 



No signal with positive controls 

• Incorrect programming of the temperature profile of the thermo cycler 

� Compare the temperature profile with the SOP. 

• 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. 

• The storage conditions for one or more product components did not 

comply with the instructions or the FTD Resp 21 had 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 in fluorimeter channel 

• 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 internal control was added during the 

extraction. 

� Make sure that your extraction method is correct. 

� A strong positive signal of a pathogen can inhibit the fluorescence 

of an internal control. 

Signals with the negative control in fluorimeter channel 

• A contamination occurred during preparation of the PCR 

� Repeat the PCR with new reagents in replicates. 

� Strictly pipette the positive controls at 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 

FTD. 

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17. Validation 

17.1. Specificity 



The BLAST (www.ncbi.nlm.nih.gov/blast) search shows that the selected primers 

and probes only detect the correspondent respiratory viruses and bacteria. The 

primer and probes of different viruses and bacteria are not cross-reacting. The 

targeted sequences for each pathogen are listed in the table below (table 5). 

Table 5: Target sequences of each pathogen. 

Pathogen Target 

FluA Matrix gene (pos1) 

H1N1 segment 6 of NA 

FluB segment 8 NS1/NEP 

Rhino 5`untranslated Region (UTR) 

Cor43 nucleocapsid protein (N) gene 



HKU nucleocapsid protein (N) gene 

Para2 hemagglutinin-neuraminidase (HN) mRNA 


Para4 fusion protein gene 


HMPV A fusion glycoprotein (F) gene 

HMPV B fusion glycoprotein (F) gene 

Mpneu adhesin P1 

HboV NP1 gene 

EV parts of domain IV and V 

PV 5`untranslated Region (UTR) 

RSVA nucleocapsid protein gene 

RSVB nucleoprotein mRNA 

AV hexon gene 

There is only one exceptional case: 

The primers and probe for the detection of Rhino also detect EV, but not the 

other way around. If you get a positive signal for Rhino and EV the patient is EV 

positive. If you just detect Rhino the patient is Rhino positive. 

- 21 -



The specificity was validated with different respiratory negative samples. These 

did not generate any positive signal with the primers and probes which are 

included in the FTD Resp 21. 

For the specificity of the FTD Resp 21 assay we analyzed a range of positive 

material containing bacterial and viral pathogens. No other bacteria or viruses 

got detected by the five PPmixes only the mentioned exceptional case. 

17.2. Sensitivity 

The analytical detection limit was tested by using a tenfold dilution series of 

plasmid DNA of known concentration (1E+09-1E+00 copies/ml) and the FTD 

Resp 21 (table 6). In addition a 10 times measuring surrounding the plasmid 

detection limit was performed (table 7). Furthermore the detection limit was 

determined by using diluted positive patient material calculated with plasmid 

DNA of known concentration (1E+09-1E+06 copies/ml) and the FTD Resp 21 

(table 6). 

Table 6: Detection limit of plasmid DNA and positive samples with using plasmid DNA as 

quantitation standard. 

PPmix Pathogen 

FluRhino 

Cor 

Para.EAV 

BoMpPf1 

RsEPA 

Positive sample Plasmid 

Detection 

limit 

(copies/ml) 

Detection 

limit 

(copies/10µl 

PCR) 

- 22 - 

Detection 

limit 

(copies/ml) 

Detection 

limit 

(copies/10µl 

PCR) 

FluA 2,05E+03 2,05E+01 1,00E+02 1,00E+00 

H1N1 2,00E+02 2,00E+00 1,00E+03 1,00E+01 

FluB 6,98E+02 6,98E+00 1,00E+03 1,00E+01 

Rhino 2,78E+02 2,78E+00 1,00E+04 1,00E+02 

Cor63 3,66E+01 3,66E-01 1,00E+03 1,00E+01 

Cor229 1,98E+03 1,98E+01 1,00E+04 1,00E+02 

Cor43 4,47E+02 4,47E+00 1,00E+02 1,00E+00 

HKU 5,17E+02 5,17E+00 1,00E+03 1,00E+01 

Para2 1,68E+02 1,68E+00 1,00E+02 1,00E+00 

Para3 5,04E+02 5,04E+00 1,00E+03 1,00E+01 

Para4 1,12E+04 1,14E+02 1,00E+04 1,00E+02 

Para1 2,37E+03 2,37E+01 1,00E+03 1,00E+01 

HMPV 5,75E+02 5,75E+00 1,00E+01 1,00E-01 

HboV 1,03E+03 1,03E+01 1,00E+03 1,00E+01 

Mpneu 2,32E+02 2,32E+00 1,00E+02 1,00E+00 

EV 4,42E+02 4,42E+00 1,00E+03 1,00E+01 

PV 8,04E+01 8,04E-01 1,00E+03 1,00E+01 

RSV 2,84E+02 2,84E+00 1,00E+03 1,00E+01 

AV 1,47E+02 1,47E+00 1,00E+03 1,00E+01



Table 7: Detection limit of plasmid DNA in 10 times measurement. Detection percentages are 

given. n.t. - not tested; neg - no target detected 

Pathogen 

plmd 10 4 

cop/ml 

plmd 10 3 

cop/ml 

- 23 - 

plmd 10 2 

cop/ml 

plmd 10 1 

cop/ml 

FluA 10/10 100% 9/10 90% 5/10 50% n.t. n.t. 

FluB 10/10 100% 7/10 70% neg neg n.t. n.t. 

Rhino 10/10 100% 10/10 100% n.t. n.t. n.t. n.t. 

H1N1 10/10 100% 9/10 90% n.t. n.t. n.t. n.t. 

Cor63 10/10 100% 10/10 100% n.t. n.t. n.t. n.t. 

Cor229 10/10 100% 9/10 90% n.t. n.t. n.t. n.t. 

Cor43 10/10 100% 10/10 100% 7/10 70% n.t. n.t. 

HKU 10/10 100% neg neg n.t. n.t. n.t. n.t. 

Para2 10/10 100% 10/10 100% 5/10 50% n.t. n.t. 

Para3 10/10 100% 10/10 100% n.t. n.t. n.t. n.t. 

Para4 10/10 100% neg neg n.t. n.t. n.t. n.t. 

Para1 10/10 100% 10/10 100% n.t. n.t. n.t. n.t. 

HMPV 10/10 100% 10/10 100% 10/10 100% 2/10 20% 

HboV 10/10 100% 10/10 100% n.t. n.t. n.t. n.t. 

Mpneu 10/10 100% 10/10 100% 7/10 70% n.t. n.t. 

AV 10/10 100% neg neg n.t. n.t. n.t. n.t. 

RSV A/B 10/10 100% neg neg n.t. n.t. n.t. n.t. 

PV 10/10 100% 8/10 80% n.t. n.t. n.t. n.t. 

EV 10/10 100% 7/10 70% n.t. n.t. n.t. n.t.

17.3. Clinical evaluation 



All clinical samples were tested with existing multiplex PCRs and the FTD Resp 

21. Only for the detection of HKU there was no reference PCR available. 

233 nasopharyngeal and pleural effusion samples were analyzed. The following 

statistic shows how many positives of each pathogen had been detected (table 

8). 

The results for the reference PCR and the FTD Resp21 were nearly comparable. 

Table 8: Statistical analysis of the clinical evaluation data for the FTD Resp 21 in comparison 

with the older version FTD Resp+. 

* 1 No older reference PCR available in FTD. 

* 2 EV and PV are dividable in the new FTD Resp 21. 

na - not analyzable 

Pathogen 

... 

Reference 

method 

FTD Resp21 

pos neg 

a c 

b d 

- 24 - 

Total Sensitivity (%) Specificity (%) 

... a/(a+c) x 100 d/(d+b) x 100

Pathogen 

FluA 

H1N1 

FluB 

Rhino 

Para2 

Para3 

Para4 

Cor43 

Cor63 

Cor229 

HKU 

HMPV 

HboV 

Para1 

Mpneu 

EV 

PV 

RSV 

AV 


Resp+ 


Flu 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 


Resp+ 

FTD Resp21plus 

pos neg 

pos 15 / 

neg / 218 

pos 20 / 


pos 9 

neg / 224 

pos 23 / 


pos 12 / 

neg / 221 

pos 12 / 

neg / 221 

pos 4 / 

neg / 229 

pos 13 / 

neg / 220 

pos 3 / 

neg / 230 

pos 6 / 

neg / 227 



- 25 - 

Total 

Sensitivity 

(%) 

Specificity 

(%) 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

nt* 1 3 230 233 na na 

pos 10 / 

neg / 223 233 100 100 

pos 3 / 

neg / 230 233 100 100 

pos 9 / 

neg / 224 233 100 100 

pos 8 / 

neg / 225 233 100 100 

pos 11* 2 / 

neg / 222 

pos 2* 2 / 

neg / 231 

pos 20 / 


pos 29 / 

neg / 204 

233 100 100 

233 100 100 

233 100 100 

233 100 100 

For detailed or further validation data like: precision, linearity, stability 

and additional diagnostic studies FTD can provide you with the 

comprehensive validation file on request!

18. Legend of symbols 



Symbol Significance 

Manufacturer 

Date Of Manufacture 

Catalogue Number 

Batch Code 

Use By 

Suffices For 

In Vitro Diagnostics Medical Device 

Temperature Limits 

Caution 

Consult Instructions For Use 

- 26 -

FTD Respiratory pathogens 21 - Mikrogen

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