Optimization of multiplex real-time RT-PCR for respiratory syncytial viruses detection
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Multiplex, real-time RT-PCR, RSV-A, RSV-B
Abstract
Abstract
Background: Multiplex real-time RT-PCR (rRT-PCR) is a fast, sensitive and specific test to detect more than one target in single PCR reaction. In this study we developed multiplex rRT-PCR for RSV-A and RSV-B since those viruses are the most common pathogen found in respiratory tract. However, in order to gain optimal reaction for RSV-A and RSV-B detection, the optimization of primers and probes specific for RSV-A and RSV B are needed.
Method: The primers and probes of multiplex rRT-PCR for RSV-A and RSV-B were selected and optimized utilizing PerlPrimer software and BLAST to analyze the secondary structures and specificity, respectively. Further testing of selected primers and probes for rRT-PCR was done using annealing temperature based on in silico analysis as mentioned above. This includes sensitivity testing with the utilization of synthesized DNA of RSV-A and RSV-B and specificity testing targeting the common viruses found in respiratory tract.
Results: The primer set and probes selected for RSV-A and RSV-B detection were specific only for RSV-A and RSV-B and showed no secondary structure. Based on primer and probe criteria for rRT-PCR such as annealing temperature, no secondary structure formed, % GC content and limit of detection, the multiplex rRT-PCR test using selected primers and probes was able to detect synthesized DNA of RSV-A and RSV-B.
Conclusion: Multiplex rRT-PCR that employing primer sets and probes targeted N gene of RSV-A and RSV-B in this study were able to be detect RSV-A and RSV-B in single PCR reaction.
Keyword: Multiplex, real-time RT-PCR, RSV-A, RSV-B
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Abstrak
Latar belakang: Multiplex real-time RT-PCR (rRT-PCR) merupakan metode yang cepat, sensitif dan spesifik untuk mendeteksi lebih dari satu target pathogen dalam satu reaksi PCR. Penelitian ini bertujuan untuk mengembangkan multiplex rRT-PCR virus RSV-A dan RSV-B yang merupakan patogen yang paling sering ditemukan di saluran pernafasan. Optimisasi dari primer dan probe dalam multiplex rRT-PCR diperlukan untuk mendapatkan reaksi yang optimal dalam deteksi virus RSV-A dan RSV-B.
Metode: Primer dan probe untuk multiplex rRT-PCR RSV-A dan RSV-B dipilih dan dioptimasi menggunakan software PerlPrimer dan BLAST untuk menganalisis adanya struktur sekunder serta spesifisitas dari primer dan probe. Uji multiplex rRT-PCR dilanjutkan berdasarkan suhu annealing berdasarkan hasil analisis menggunakan PerlPrimer. Uji sensitifitas dilakukan dengan menggunakan DNA sintetis dari RSV-A dan RSV-B dan uji spesifisitas dilakukan dengan mengetes primer dan probe terhadap virus-virus lain yang umumnya ditemukan di saluran pernafasan.
Hasil: Primer dan probe yang dikembangkan pada penelitian ini tidak membentuk struktur sekunder dan spesifik mengamplifikasi hanya RSV-A dan RSV-B. Berdasarkan kriteria primer dan probe untuk digunakan dalam rRT-PCR yaitu suhu annealing, tidak adanya pembentukan struktur sekunder, % GC content serta detection limit, uji multiplex rRT-PCR yang dikembangkan pada penelitian ini mampu mendeteksi DNA sintetis RSV-A dan RSV-B.
Kesimpulan: Multiplex rRT-PCR dengan menggunakan primer dan probe untuk RSV-A dan RSV-B dapat mendeteksi RSV-A dan RSV-B dalam satu reaksi PCR.
Kata kunci: multiplex, real-time RT-PCR, RSV-A, RSV-B
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References
Olofsson S, Brittain-Long R, Andersson LM, Westin J, Lindh M. PCR for detection of respiratory viruses: seasonal variations of virus infections. Expert Rev Anti Infect Ther. Aug 2011;9(8):615-26.
Appak O, Duman M, Belet N, Sayiner AA. Viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients. J Med Virol. May 2019;91(5):731-7.
Wertheim HFL, Nadjm B, Thomas S, et al. Viral and atypical bacterial aetiologies of infection in hospitalised patients admitted with clinical suspicion of influenza in Thailand, Vietnam and Indonesia. Influenza Other Respir Viruses. Nov 2015;9(6):315-22.
Nair H, Nokes DJ, Gessner BD, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet. May 1 2010;375(9725):1545-55.
Sonego M, Pellegrin MC, Becker G, Lazzerini M. Risk factors for mortality from acute lower respiratory infections (ALRI) in children under five years of age in low and middle-income countries: a systematic review and meta-analysis of observational studies. PLoS One. 2015;10(1):e0116380.
Walker CLF, Rudan I, Liu L, et al. Global burden of childhood pneumonia and diarrhoea. Lancet. Apr 20 2013;381(9875):1405-16.
Ruuskanen O, Lahti E, Jennings LC, Murdoch DR. Viral pneumonia. Lancet. Apr 9 2011;377(9773):1264-75.
Zar HJ, Ferkol TW. The global burden of respiratory disease-impact on child health. Pediatr Pulmonol. May 2014;49(5):430-4.
Jartti T, Soderlund-Venermo M, Hedman K, Ruuskanen O, Makela MJ. New molecular virus detection methods and their clinical value in lower respiratory tract infections in children. Paediatr Respir Rev. Mar 2013;14(1):38-45.
Zimmerman RK, Rinaldo CR, Nowalk MP, et al. Influenza and other respiratory virus infections in outpatients with medically attended acute respiratory infection during the 2011-12 influenza season. Influenza Other Respir Viruses. Jul 2014;8(4):397-405.
de-Paris F, Beck C, de Souza Nunes L, et al. Evaluation of respiratory syncytial virus group A and B genotypes among nosocomial and community-acquired pediatric infections in Southern Brazil. Virology journal. Feb 24 2014;11:36.
Munoz-Escalante JC, Comas-Garcia A, Bernal-Silva S, Noyola DE. Respiratory syncytial virus B sequence analysis reveals a novel early genotype. Scientific reports. Feb 10 2021;11(1):3452.
Brittain-Long R, Westin J, Olofsson S, Lindh M, Andersson LM. Access to a polymerase chain reaction assay method targeting 13 respiratory viruses can reduce antibiotics: a randomised, controlled trial. BMC Med. Apr 26 2011;9:44.
van Elden LJ, van Loon AM, van der Beek A, et al. Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults. J Clin Microbiol. Sep 2003;41(9):4378-81.
Hall TA. BioEdit: A user-friendly biologycal sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95-98.
Marshall O. Graphical design of primers with PerlPrimer. Methods Mol Biol. 2007;402:403-14.
Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics. Oct 12 2004;20(15):2471-2.
Mahony JB, Petrich A, Smieja M. Molecular diagnosis of respiratory virus infections. Critical reviews in clinical laboratory sciences. Sep-Dec 2011;48(5-6):217-49.
Artika IM, Wiyatno A, Ma'roef CN. Pathogenic viruses: molecular detection and characterization. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. Jul 2020;81:104215.
Innis MA, Gelfand DH. Optimization of PCRs. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols a guide to methods and applications. San Diego California 92101: Academic Press, Inc.; 1990:3-12.
Kampke T. The Reference Point Method in Primer Design. In: Yuryev A, ed. PCR Primer Design. Totowa, New Jersey 07512: Hamuna Press; 2007:75-92.
Navarro E, Serrano-Heras G, Castano MJ, Solera J. Real-time PCR detection chemistry. Clinica chimica acta; international journal of clinical chemistry. Jan 15 2015;439:231-50.
Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical chemistry. Apr 2009;55(4):611-22.