Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
2015; Elsevier BV; Volume: 17; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2015.01.005
ISSN1943-7811
AutoresAlicia Edin, Susanne Granholm, Satu Koskiniemi, Annika Allard, Anders Sjöstedt, Anders Johansson,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoCommunity-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were 95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens. Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were 95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens. Community-acquired pneumonia (CAP) represents a major challenge for institutions of rational medical treatment because of its diverse cause. Viruses and bacteria may present with similar symptoms, making them difficult to distinguish clinically.1Huijskens E.G. Koopmans M. Palmen F.M. van Erkel A.J. Mulder P.G. Rossen J.W. The value of signs and symptoms in differentiating between bacterial, viral and mixed aetiology in patients with community-acquired pneumonia.J Med Microbiol. 2014; 63: 441-452Crossref PubMed Scopus (59) Google Scholar When physicians lack information about the causative agent for their decision on appropriate treatment, it leads to increased use of ecologically unfavorable broad-spectrum antibiotics.2Caliendo A.M. Gilbert D.N. Ginocchio C.C. Hanson K.E. May L. Quinn T.C. Tenover F.C. Alland D. Blaschke A.J. Bonomo R.A. Carroll K.C. Ferraro M.J. Hirschhorn L.R. Joseph W.P. Karchmer T. MacIntyre A.T. Reller L.B. Jackson A.F. Infectious Diseases Society of America (IDSA)Better tests, better care: improved diagnostics for infectious diseases.Clin Infect Dis. 2013; 57: S139-S170Crossref PubMed Scopus (464) Google Scholar To provide appropriate and targeted treatment, physicians need diagnostic methods that are rapid, sensitive, and specific, the combination of which current standard diagnostic methods lack. Although present viral diagnostics mostly rely on nucleic acid detection, the properties of traditional cultivation techniques for bacteria limit the possibilities to develop a rapid method for detection of all tentative causes. Thus, a number of recent studies have aimed to develop molecular methods to detect bacterial and viral pathogens. To overcome the limitations of cultivation, quantitative real-time PCR (qPCR) assays were implemented because they allow rapid detection and are generally applicable. The method was implemented for common bacterial pathogens such as Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae, Moraxella catarrhalis, Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae.3Welti M. Jaton K. Altwegg M. Sahli R. Wenger A. Bille J. Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.Diagn Microbiol Infect Dis. 2003; 45: 85-95Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 4Gullsby K. Storm M. Bondeson K. Simultaneous detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by use of molecular beacons in a duplex real-time PCR.J Clin Microbiol. 2008; 46: 727-731Crossref PubMed Scopus (33) Google Scholar, 5Abdeldaim G.M. Strålin K. Korsgaard J. Blomberg J. Welinder-Olsson C. Herrmann B. 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A recent evaluation of two available systems found that, although one clinically relevant pathogen could readily be detected, there were performance limitations when two pathogens were present in a specimen.13Driscoll A.J. Karron R.A. Bhat N. Thumar B. Kodani M. Fields B.S. Whitney C.G. Levine O.S. O'Brien K.L. Murdoch D.R. Evaluation of fast-track diagnostics and TaqMan array card real-time PCR assays for the detection of respiratory pathogens.J Microbiol Methods. 2014; 107: 222-226Crossref PubMed Scopus (25) Google Scholar Because of the rapid turnaround time and a low limit of detection, implementing nucleic acid-based detection methods for CAP pathogens may shorten the time to diagnosis and increase sensitivity. Standard cultivation methods provide limited sensitivity for important causes, including H. influenzae, probably because of the fastidious nature of the bacterium, and even more so for S. pneumoniae. Because of autolytic behavior, the latter seems to survive poorly during both transport and after cultivation.14Jedrzejas M.J. Pneumococcal virulence factors: structure and function.Microbiol Mol Biol Rev. 2001; 65: 187-207Crossref PubMed Scopus (384) Google Scholar To fully take advantage of the molecular methods' sensitivity and specificity, target genes must be chosen with care. Capitalizing on previous work that validated suitable targets for several respiratory pathogens, we have developed a qPCR assay for causative diagnosis of CAP.3Welti M. Jaton K. Altwegg M. Sahli R. Wenger A. Bille J. Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.Diagn Microbiol Infect Dis. 2003; 45: 85-95Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 12Brittain-Long R. Nord S. Olofsson S. Westin J. Anderson L.M. Lindh M. Multiplex real-time PCR for detection of respiratory tract infections.J Clin Virol. 2008; 41: 53-56Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 15Heiniger N. Spaniol V. Troller R. Vischer M. Aebi C. A reservoir of Moraxella catarrhalis in human pharyngeal lymphoid tissue.J Infect Dis. 2007; 196: 1080-1087Crossref PubMed Scopus (60) Google Scholar, 16Liu D. Hollingshead S. Swiatlo E. Lawrence M.L. Austin F.W. Rapid identification of Streptococcus pyogenes with PCR primers from a putative transcriptional regulator gene.Res Microbiol. 2005; 156: 564-567Crossref PubMed Scopus (36) Google Scholar, 17Strålin K. Bäckman A. Holmberg H. Fredlund H. Olcén P. Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples.APMIS. 2005; 113: 99-111Crossref PubMed Scopus (69) Google Scholar, 18Abdeldaim G.M. Strålin K. Kirsebom L.A. Olcén P. Blomberg J. Herrmann B. Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.Diagn Microbiol Infect Dis. 2009; 64: 366-373Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 19Martinon A. Cronin U.P. Wilkinson M.G. Development of defined microbial population standards using fluorescence activated cell sorting for the absolute quantification of S. aureus using real-time PCR.Mol Biotechnol. 2012; 50: 62-71Crossref PubMed Scopus (6) Google Scholar, 20Fosheim G.E. Nicholson A.C. Albrecht V.S. Limbago B.M. Multiplex real-time PCR assay for detection of methicillin-resistant Staphylococcus aureus and associated toxin genes.J Clin Microbiol. 2011; 49: 3071-3073Crossref PubMed Scopus (36) Google Scholar, 21Templeton K.E. Scheltinga S.A. Beersma M.F. Kroes A.C. Claas E.C. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza a and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4.J Clin Microbiol. 2004; 42: 1564-1569Crossref PubMed Scopus (410) Google Scholar, 22Watzinger F. Suda M. Preuner S. Baumgartinger R. Ebner K. Baskova L. Niesters H.G. Lawitschka A. Lion T. Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed.J Clin Microbiol. 2004; 42: 5189-5198Crossref PubMed Scopus (270) Google Scholar, 23Ward C.L. Dempsey M.H. Ring C.J. Kempson R.E. Zhang L. Gor D. Snowden B.W. Tisdale M. Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement.J Clin Virol. 2004; 29: 179-188Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar We have emphasized high technical performance, including the ability to detect co-infections when multiple pathogens are present at different concentrations. Here, we present a multiplex, qPCR method for simultaneous detection of eight bacterial and six viral pathogens with relevance for diagnosis of CAP. This work was conducted at the maximum possible extent according to the guidelines on minimum information for publication of qPCR experiments.24Bustin S.A. Benes V. Garson J.A. Hellemans J. Huggett J. Kubista M. Mueller R. Nolan T. Pfaffl M.W. Shipley G.L. Vandesompele J. Wittwer C.T. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.Clin Chem. 2009; 55: 611-622Crossref PubMed Scopus (11572) Google Scholar Sputum specimens were homogenized with equal volume N-acetyl cysteine solution as described previously,25Tebbutt G.M. Coleman D.J. Evaluation of some methods for the laboratory examination of sputum.J Clin Pathol. 1978; 31: 724-729Crossref PubMed Scopus (11) Google Scholar before extraction of nucleic acids. Automated DNA/RNA extraction by the Nordiag Arrow instrument and the Viral NA kit (DiaSorin, Saluggia VC, Italy) was used for all clinical specimens and viral strains with a sample volume of 550 μL and an elution volume of 100 μL. The eluate of clinical samples was then diluted with 100 μL of nuclease-free water (Ambion, Life Technologies, Carlsbad, CA) before being stored at −80°C until analysis. DNA of bacterial strains was extracted by a previously described method,26Ibrahim A. Norlander L. Macellaro A. Sjöstedt A. Specific detection of Coxiella burnetii through partial amplification of 23S rDNA.Eur J Epidemiol. 1997; 13: 329-334Crossref PubMed Scopus (19) Google Scholar based on binding of DNA to uniform glass beads, before being stored at −20°C until analysis. Plasmid DNA was extracted by the QIAprep Spin Miniprep Kit (Qiagen Inc., Valencia, CA), according to the manufacturer's instructions. Linear plasmids with inserts of the target sequences were used as positive controls for all assays. Plasmids were ordered from Eurofins MWG Operon (Ebersberg, Germany), except the plasmid used for C. pneumoniae that was an in-house construct. Plasmids were transformed into One Shot TOP10 Electrocom Escherichia coli (Invitrogen, Life Technologies, Carlsbad, CA); cells were plated on kanamycin-containing Luria Broth-agar plates. Single colonies were used for DNA extraction as described in DNA and RNA Extraction, and the concentration was measured in duplicates with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). A positive control solution of 2 × 104 plasmid copies per mL that corresponded to 100 plasmid copies per 25 μL of PCR reaction was prepared. Primer and probe sequences for L. pneumophila, M. pneumoniae, M. catarrhalis, C. pneumoniae, S. aureus, PIV type 1 (PIV1), PIV2, RSV, and influenza A1 and B were obtained from previous publications,3Welti M. Jaton K. Altwegg M. Sahli R. Wenger A. Bille J. Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.Diagn Microbiol Infect Dis. 2003; 45: 85-95Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 12Brittain-Long R. Nord S. Olofsson S. Westin J. Anderson L.M. Lindh M. Multiplex real-time PCR for detection of respiratory tract infections.J Clin Virol. 2008; 41: 53-56Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 15Heiniger N. Spaniol V. Troller R. Vischer M. Aebi C. A reservoir of Moraxella catarrhalis in human pharyngeal lymphoid tissue.J Infect Dis. 2007; 196: 1080-1087Crossref PubMed Scopus (60) Google Scholar, 20Fosheim G.E. Nicholson A.C. Albrecht V.S. Limbago B.M. Multiplex real-time PCR assay for detection of methicillin-resistant Staphylococcus aureus and associated toxin genes.J Clin Microbiol. 2011; 49: 3071-3073Crossref PubMed Scopus (36) Google Scholar, 22Watzinger F. Suda M. Preuner S. Baumgartinger R. Ebner K. Baskova L. Niesters H.G. Lawitschka A. Lion T. Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed.J Clin Microbiol. 2004; 42: 5189-5198Crossref PubMed Scopus (270) Google Scholar and PCR conditions were optimized for duplex reactions (Table 1). A new influenza A H3 strain with point mutations in the target sequence of primers and probes of the influenza A1 assay appeared during the course of our work, and our qPCR assay was therefore updated according to the recommendations of the Public Health Agency of Sweden (M. Brytting, unpublished data) to include an assay-denoted influenza A2. Primers and probes for H. influenzae and S. pneumoniae were already established as uniplex qPCR assays for analysis of spinal fluid at our laboratory but not published before. Primers and probes for S. pyogenes were constructed with the Primer Express version 3.0 (Applied Biosystems, Life Technologies, Carlsbad, CA). All probes used the hydrolysis technology with black hole quenchers and fluorophores 6-carboxyfluorescein or VIC (Applied Biosystems). The TaqMan Fast Advanced Master Mix for DNA detection and the TaqMan Fast Virus 1-step Master Mix for RNA detection were used for bacteria and viruses, respectively (Applied Biosystems). Total reaction volume was 25 μL, consisting of 12.5/6.25 μL of 2× DNA/4× RNA Master Mix, primers, probes, nuclease-free water, and 5 μL of template or clinical specimen. All runs included triplicates of no-template controls and duplicates of positive controls. Amplification, detection, and analysis were performed by a 7900HT Fast Real-Time PCR System (Applied Biosystems). The thermal profile was 5 minutes at 50°C, 20 seconds at 95°C, and subsequently 45 cycles of 15 seconds at 95°C and 60 seconds at 60°C.Table 1Primers and ProbesPrimer/probe target (gene or protein name)SequenceReporter/quencherConcentration, nmol/LReferenceDuplex nameLegionella pneumophila (mip)Forward: 5′-AAAGGCATGCAAGACGCTATG-3′1003Welti M. Jaton K. Altwegg M. Sahli R. Wenger A. Bille J. Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.Diagn Microbiol Infect Dis. 2003; 45: 85-95Abstract Full Text Full Text PDF PubMed Scopus (166) Google ScholarAReverse: 5′-TGTTAAGAACGTCTTTCATTTGCTG-3′100Probe: 5′-TGGCGCTCAATTGGCTTTAACCGA-3′VIC-MGBNFQ250Mycoplasma pneumonia (P1)Forward: 5′-GGAATCCCAATGCACAAGAACA-3′60012Brittain-Long R. Nord S. Olofsson S. Westin J. Anderson L.M. Lindh M. Multiplex real-time PCR for detection of respiratory tract infections.J Clin Virol. 2008; 41: 53-56Abstract Full Text Full Text PDF PubMed Scopus (180) Google ScholarReverse: 5′-GCTTTGGTCAACACATCAACCTT-3′300Probe: 5′-GCCTTGAAGGCTGGGTTTGCGCTA-3′6FAM-MGBNFQ250Streptococcus pneumoniae (lytA)Forward: 5′-AACTCTTACGCAATCTAGCAGATGAA-3′300This studyBReverse: 5′-CGTGCAATACTCGTGCGTTTTA-3′300Probe: 5′-CCGAAAACGCTTGATACA-3′6FAM-MGBNFQ250Moraxella catarrhalis (uspA1)Forward: 5′-GTCAAACAGCTGGAGGTATTGC-3′30015Heiniger N. Spaniol V. Troller R. Vischer M. Aebi C. A reservoir of Moraxella catarrhalis in human pharyngeal lymphoid tissue.J Infect Dis. 2007; 196: 1080-1087Crossref PubMed Scopus (60) Google ScholarReverse: 5′-GACATGATGCTCACCTGCTCTA-3′600Probe: 5′-ATCGCAATTGCAACTTT-3′VIC-MGBNFQ200Chlamydophila pneumonia (momp)Forward: 5′-CAAGGGCTATAAAGGCGTTGCT-3′20012Brittain-Long R. Nord S. Olofsson S. Westin J. Anderson L.M. Lindh M. Multiplex real-time PCR for detection of respiratory tract infections.J Clin Virol. 2008; 41: 53-56Abstract Full Text Full Text PDF PubMed Scopus (180) Google ScholarCReverse: 5′-ATGGTCGCAGACTTTGTTCCA-3′200Probe: 5′-TCCCCTTGCCAACAGACGCTGG-3′VIC-MGBNFQ250Staphylococcus aureus (nuc)Forward: 5′-AAATTACATAAAGAACCTGCGACA-3′30020Fosheim G.E. Nicholson A.C. Albrecht V.S. Limbago B.M. Multiplex real-time PCR assay for detection of methicillin-resistant Staphylococcus aureus and associated toxin genes.J Clin Microbiol. 2011; 49: 3071-3073Crossref PubMed Scopus (36) Google ScholarReverse: 5′-GAATGTCATTGGTTGACCTTTGTA-3′600Probe: 5′-AATTTAACCGTATCACCATCAATCGCTTT-3′6FAM-MGBNFQ250Streptococcus pyogenes grp A (SPy_1285)Forward: 5′-CTCGACAAGTCCTCAATCAAACC-3′200This studyDReverse: 5′-ATGAGTTGCGGAAATTTGAGGTA-3′200Probe: 5′-CATAGAGAATTTATAACCGCACTC-3′6FAM-MGBNFQ250Haemophilus influenza (P6)Forward: 5′-CTTGGTCTCCATACTTAACTAAATAAAAAACTC-3′400This studyReverse: 5′-GAACTACAAGCCGCTAATGCAG-3′400Probe: 5′-CATTATTAGTTGCAGGTTCT-3′VIC-MGBNFQ250Parainfluenza virus 1 (HN)Forward: 5′-GTTGTCAATGTCTTAATTCGTATCAATAATT-3′30022Watzinger F. Suda M. Preuner S. Baumgartinger R. Ebner K. Baskova L. Niesters H.G. Lawitschka A. Lion T. Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed.J Clin Microbiol. 2004; 42: 5189-5198Crossref PubMed Scopus (270) Google ScholarEReverse: 5′-GTAGCCTMCCTTCGGCACCTAA-3′300Probe: 5′-TAGGCCAAAGATTGTTGTCGAGACTATTCCAAGCATTTCCAATCTTCAGGACTATGA-3′6FAM-MGBNFQ150Parainfluenza virus 2 (HN)Forward: 5′-GCATTTCCAATCTTCAGGACTATGA-3′30022Watzinger F. Suda M. Preuner S. Baumgartinger R. Ebner K. Baskova L. Niesters H.G. Lawitschka A. Lion T. Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed.J Clin Microbiol. 2004; 42: 5189-5198Crossref PubMed Scopus (270) Google ScholarReverse: 5′-ACCTCCTGGTATAGCAGTGACTGAAC-3′300Probe: 5′-CCATTTACCTAAGTGATGGAATCAATCGCAAA-3′VIC-MGBNFQ250Parainfluenza virus 3 (HN)Forward: 5′-AAATGATCTGATTTATGCTTATACCTC-3′300LP Nielsen, unpublished dataFReverse: 5′-TCAGGTACCAAGTCTGAGTTTACA-3′300CopenhagenProbe: 5′-CGAGGTTGYCAGGATATAGGAAAATCA-3′6FAM-MGBNFQ250Respiratory syncytial virus (M)Forward: 5′-GCAAATATGGAAACATACGTGAACA-3′20012Brittain-Long R. Nord S. Olofsson S. Westin J. Anderson L.M. Lindh M. Multiplex real-time PCR for detection of respiratory tract infections.J Clin Virol. 2008; 41: 53-56Abstract Full Text Full Text PDF PubMed Scopus (180) Google ScholarReverse: 5′-GCACCCATATTGTWAGTGATGCA-3′200Probe: 5′-CTTCACGAAGGCTCCACATACACAGCWG-3′VIC-MGBNFQ150Influenza A 1 (M1)∗For the detection of influenza A, two different primer pairs were used during the study because a new influenza A strain emerged, and we adjusted our assay accordingly.Forward: 5′-AAGACCAATYCTGTCACCTCTGA-3′60012Brittain-Long R. Nord S. Olofsson S. Westin J. Anderson L.M. Lindh M. Multiplex real-time PCR for detection of respiratory tract infections.J Clin Virol. 2008; 41: 53-56Abstract Full Text Full Text PDF PubMed Scopus (180) Google ScholarGReverse: 5′-CAAAGCGTCTACGCTGCAGTCC-3′300Probe: 5′-TTTGTGTTCACGCTCACCGT-3′VIC-MGBNFQ250Influenza A 2 (Segment 7/M1)∗For the detection of influenza A, two different primer pairs were used during the study because a new influenza A strain emerged, and we adjusted our assay accordingly.Forward 1: 5′-AGGACCYCTGTCACCTCTGA-3′300M Brytting, unpublished dataForward 2: 5′-CAAGACCAATCTTGTCACTCTGA-3′300Public Health Agency of SwedenReverse 1: 5′-TCTCGCTCACTGGGCA-3′300Reverse 2: 5′-TCCTCGCTCACTTGGCA-3′300Probe 1: 5′-TTGTGTTCACGCTCACC-3′6FAM-MGBNFQ112Probe 2: 5′-TTTGTTTTCACGCTCACCG-3′6FAM-MGBNFQ112Probe 3: 5′-TTTGTATTCACGCTCACCG-3′6FAM-MGBNFQ112Influenza B (HA)Forward: 5′-AAATACGGTGGATTAAAYAAAAGCAA-3′60012Brittain-Long R. Nord S. Olofsson S. Westin J. Anderson L.M. Lindh M. Multiplex real-time PCR for detection of respiratory tract infections.J Clin Virol. 2008; 41: 53-56Abstract Full Text Full Text PDF PubMed Scopus (180) Google ScholarReverse: 5′-CCAGCAATAGCTCCGAAGAAA-3′600Probe: 5′-CACCCATATTGGGCAATTTCCTATGGC-3′VIC-MGBNFQ2506FAM, 6-carboxyfluorescein.Bold indicates the use of a primer mix to target two different bases.∗ For the detection of influenza A, two different primer pairs were used during the study because a new influenza A strain emerged, and we adjusted our assay accordingly. Open table in a new tab 6FAM, 6-carboxyfluorescein. Bold indicates the use of a primer mix to target two different bases. Duplex qPCR assays were combined as described in Table 1. Analytical specificity of primers and probes was tested bioinformatically and experimentally with primer BLAST27Ye J. Coulouris G. Zaretskaya I. Cutcutache I. Rozen S. Madden T.L. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.BMC Bioinformatics. 2012; 13: 134Crossref PubMed Scopus (4051) Google Scholar and a test panel of 34 reference strains (Supplemental Table S1), respectively. Limit of detection was assessed as recommended,28Codex Committee on Methods of Analysis and Sampling. 27th Session of the European Community Comments on items 4, 7 and 9, 2006 May 15-19. Budapest, Hungary. pp 1–6Google Scholar by serial dilution of linear plasmid by using six replicates with concentrations of 20, 10, 5, 2, 1, and 0.1 copies per reaction, respectively. The ability of the duplex assay to simultaneously detect both agents targeted was tested by keeping one of the agents at high concentration (104 copies per reaction) while testing for the other agent. For PIV1 and PIV2 equal concentrations were used. The limit of detection was defined as the concentration at which all six replicates were detected, for theoretical reasons five copies per reactions was set as detection limit. An experiment was approved only if zero to one of the six replicates in the 0.1 dilution were positive. Efficiency was tested by dilution series of plasmid standards, ranging from approximately 1 to 105 copies per reaction, with equal concentrations of both targets of the duplex reaction. The analyses were performed in duplicates or triplicates, and detection of at least five dilution steps was required for an approved result. Standard curves were generated by plotting the quantification cycle (Cq) value against 10-logarithm of the concentration. Linearity (R2 value) and slope of the standard curve was calculated with the data analysis program SDS version 2.3 (Applied Biosystems). Reproducibility of assays were assessed by comparing positive control results from 26 runs in two separate analysis batches performed >6 months apart by two persons with the use of separate plasmid preparations and dilution series. Relative SD of the Cq values was calculated as a measure of stability. In addition, reproducibility was assessed with clinical specimens spiked with low (n = 5) or high (n = 5) concentration of target DNA or RNA and by using separate qPCR machines in five consecutive runs on three different days. The relative SDs of the Cq values were calculated as a measure of intrarun variability. The presence and frequency of PCR inhibition was investigated by analysis of 22 clinical nasopharyngeal aspirates (NpAs) and 38 sputum specimens, as previously described.29Gallup J.M. Sow F.B. Van Geelen A. Ackermann M.R. SPUD qPCR assay confirms PREXCEL-Q softwares ability to avoid qPCR inhibition.Curr Issues Mol Biol. 2010; 12: 129-134PubMed Google Scholar Specimens were spiked with 100 copies per reaction with each of the plasmid standards, and the results were normalized to the mean of the 100 copies per reaction positive controls from the same run. Absence of signal or a normalized Cq value >2.5 SD above the mean of the positive controls was considered as inhibition. To verify that the method could detect viruses in sputum, sputum specimens each with a volume of 350 μL were spiked with100 μL from a NpA specimen positive for either RSV or influenza A before nucleic acid extraction and qPCR assay analysis were performed as described in the sections DNA and RNA Extraction and Primers Probes and qPCR Conditions, respectively. Lower respiratory tract specimens (expectorated or induced sputum) and upper respiratory tract specimens (NpA) sent for respiratory infection diagnostics to the Clinical Microbiology Laboratory (Norrlands universitetssjukhus, Umeå, Sweden) were consecutively included in the evaluation of the qPCR assay. Sputum specimens with an original volume of >0.5 mL, >25 leucocytes, and <10 epithelial cells per 100× power field as determined by Gram staining were included. Sputum specimens were split into one portion for standard tests and another for storage at −80°C until used for evaluation by the qPCR assay. Before culture, specimens were treated with a 1:1 volume of N-acetyl cysteine and diluted 1:100 and 1:10,000 in phosphate-buffered saline. The qPCR assay was run in small batches in parallel with standard microbiological tests, but its results were not included in the laboratory reports sent to the clinicians. The qPCR assay results were compared with all available standard test results that corresponded to the day of sampling and to the specimen type, NpA, nasopharyngeal swab, or sputum. The sta
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