FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN

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, ViiA7, 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

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 2 -

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 ................................................................................. 22

14. SETUP ON THE ABI® 7500 ........................................................................ 23

15. INTERPRETATION OF RESULTS ................................................................ 26

16. TROUBLESHOOTING ................................................................................ 28

17. VALIDATION ............................................................................................ 29

18. LEGEND OF SYMBOLS ............................................................................... 29

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 3 -

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 4 -

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 5 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 6 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 7 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 8 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 9 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 10 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 11 -

FTD Respiratory pathogens 33

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

ParaEAV PP Primer/probe mix for HPIV2-4 & IC (EAV)

1 x 48 µl 2 x 48 µl

BoMpPf1 PP Primer/probe mix for HBoV, Mpneu, HPIV1 & HMPVA/B

1 x 48 µl 2 x 48 µl

RsEPA PP Primer/probe mix for HRSVA & B, HAdV, EV & HPeV

1 x 48 µl 2 x 48 µl

RespBac PP Primer/probe mix for Saur, Spneu, Cpneu & HIB

1 x 48 µl 2 x 48 µl

KLePSa PP Primer/probe mix for Kpneu, Lpneu/Llong, PCP & Salm 1 x 48 µl 2 x 48 µl

MoBoCH PP Primer/probe mix for Morax, Bord, FLUC & Haeinf 1 x 48 µl 2 x 48 µl

Resp21 PC

Positive control: plasmid pool for FLUA, FLUB, H1N1swl, RV, Cor63, Cor229, Cor43, HKU, HPIV1-4, HMPV, HBoV, Mpneu, HRSV, HAdV, EV & HPeV

1 x 750 µl 2 x 750 µl

Resp33 PC2

Positive control: plasmid pool for use with PPmixes 6-8. This pool includes Saur, Spneu, Cpneu, HIB, Kpneu, Lpneu/Llong, PCP, Salm, Morax, Bord, FLUC and Haeinf.

1 x 450 µl 2 x 450 µl

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 12 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 13 -

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 14 -

FTD Respiratory pathogens 33

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 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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 15 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 16 -

FTD Respiratory pathogens 33

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 17 -

FTD Respiratory pathogens 33

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

FTD-2P.3-

32/64

Buffer 12.5 µl 187.5 µl 400 µl 800 µl

PPmix 1.5 µl 22.5 µl 48 µl 96 µl

Enzyme 1 µl 15 µl 32 µl 64 µl

Total 15 µl 225 µl 480 µl 960 µl

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 18 -

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 19 -

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)

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 20 -

FTD Respiratory pathogens 33

12. Programming of the thermocycler

Pay particular attention to the settings for the detectors:

Table 4: Settings of the detectors.

PP mix Pathogen Dye Detection wavelength

(nm)*

FluRhino PP

FLUA green 520

RV yellow 550

FLUB orange 610

H1N1 red 670

COR PP

Cor 229 green 520

Cor 63 yellow 550

HKU1 orange 610

Cor 43 red 670

ParaEAV PP

HPIV3 green 520

HPIV2 yellow 550

HPIV4 orange 610

IC (EAV) red 670

BoMpPf1 PP

HPIV1 green 520

HMPV A/B yellow 550

HBoV orange 610

Mpneu red 670

RsEPA PP

HRSVA/B green 520

HPeV yellow 550

EV orange 610

HAdV red 670

RespBac PP

S.aur green 520

C.pneu yellow 550

HiB orange 610

S.pneu red 670

KLePSa PP

PCP green 520

Lpneu/Llong yellow 550

K.pneu orange 610

Salm red 670

MoBoCH PP

Morax green 520

FLUC yellow 550

Bord orange 610

Haeinf red 670

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

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 21 -

FTD Respiratory pathogens 33

PCR programme: Fast-track mastermix 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.

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. The reagents for FTD color compensation are supplied by FTD. 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 (Fast-track diagnostics). If you want to use different enzymes please refer to FTD.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 22 -

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 23 -

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”.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 24 -

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].

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 25 -

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 26 -

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!

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 27 -

FTD Respiratory pathogens 33

Table 5: Possible results with FTD Respiratory pathogens 33. Pos= positive; empty= negative

PP mix Pathogen signal in

green channel

signal in yellow

channel

signal in orange channel

signal in red

channel

FluRhino PP

FLUA POS

RV POS

FLUB POS

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.

COR PP

Cor229 POS

Cor63 POS

HKU1 POS

Cor43 POS

ParaEAV PP

HPIV3 POS

HPIV2 POS

HPIV4 POS

IC (EAV) POS

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

BoMpPf1 PP

HPIV1 POS

HMPV A/B POS

HBoV POS

Mpneu POS

RsEPA PP

RSVA&B POS

HPeV POS

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.

RespBac PP

Saur POS

Cpneu POS

HIB POS

Spneu POS

KLePSa PP

PCP POS

Lpneu/Llong POS

Kpneu POS

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.

MoBoCH PP

Morax POS

FLUC POS

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 28 -

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.

FTD 2P.3 – 32_64 – MANUAL- v3 – 2016_04 EN - 29 -

FTD Respiratory pathogens 33

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