Document Type : Original Research
Dept. of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Dept. of Medical Microbiology, Kerman University of Medical Sciences, Kerman, Iran
Background and objective:Millions of people in developing countries lose their lives due to acute respiratory infections, such as Influenza A & B and Adeno viruses. Given the importance of rapid identification of the virus, in this study the researchers attempted to design a method that enables detection of influenza A, B, and adenoviruses, quickly and simultaneously. The Multiplex RT PCR method was the preferred method for the detection of influenza A, B, and adenoviruses in clinical specimens because it is rapid, sensitive, specific, and more cost-effective than alternative methods
Methods:After collecting samples from patients with respiratory disease, virus genome was extracted, then Monoplex PCR was used on positive samples and Multiplex RT-PCR on clinical specimens. Finally, by comparing the bands of these samples, the type of virus in the clinical samples was determined.
Results:Performing Multiplex RT-PCR on 50 samples of respiratory tract led to following results; flu A: 12.5%, fluB: 50%, adeno: 27.5%, negative: 7.5%, and 2.5% contamination.
Conclusion: Reverse transcription-multiplex Polymerase Chain Reaction (PCR) technique, a rapid diagnostic tool, has potential for high-throughput testing. This method has a significant advantage, which provides simultaneous amplification of numerous viruses in a single reaction. This study concentrates on multiplex molecular technologies and their clinical application for the detection and quantification of respiratory pathogens. The improvement in diagnostic testing for viral respiratory pathogens effects patient management, and leads to more cost-effective delivery of care. It limits unnecessary antibiotic use and improves clinical management by use of suitable treatment.
- Piewbang C, Rungsipipat A, Poovorawan Y, Techangamsuwan S. Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs. J Vet Med Sci 2016;16-0342.
- Long SS. Multiplex PCR testing during respiratory infections: good for the patient, good for infection prevention and antimicrobial stewardship. J Pediatr 2016;173:1-3.
- Nikonova AA, Uspenskaia ES, Lobodanov SA, Oksanich AS, Gorbalenia AE, Claas EC, et al. [Use of multiplex PCR method with real-time detection for differential diagnosis of respiratory viral infections]. Zh Mikrobiol, Epidemiol, Immunobiol 2008(1);67-70.
- Nickbakhsh S, Thorburn F, Von Wissmann B, Mcmenamin J, Gunson RN, Murcia PR. Extensive multiplex PCR diagnostics reveal new insights into the epidemiology of viral respiratory infections. Epidemiol Infect 2016;144(10):2064-76.
- Pabbaraju K, Wong S, Lee B, Tellier R, Fonseca K, Louie M, et al. Comparison of a singleplex real-time RT-PCR assay and multiplex respiratory viral panel assay for detection of influenza "A" in respiratory specimens. Influenza Other Respir Viruses 2011;5(2):99-103.
- Parker J, Fowler N, Walmsley ML, Schmidt T, Scharrer J, Kowaleski J, et al. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses. PLoS One 2015;10(11):e0143164.
- Pozzetto B, Grattard F, Pillet S. Multiplex PCR theranostics of severe respiratory infections. Expert Review of Anti Infective Therapy 2010;8(3):251-3.
- Rappo U, Schuetz AN, Jenkins SG, Calfee DP, Walsh TJ, Wells MT, et al. Impact of Early Detection of Respiratory Viruses by Multiplex PCR Assay on Clinical Outcomes in Adult Patients. J Clin Microbiol 2016;54(12):JCM.00549-16.
- Beck ET, Jurgens LA, Kehl SC, Bose ME, Patitucci T, Lague E, et al. Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin. J Mol Diagn 2010;12(1):74-81.
- Iha Y, Kinjo T, Parrott G, Higa F, Mori H, Fugita J. Comparative epidemiology of influenza A and B viral infection in a subtropical region: a 7-year surveillance in Okinawa, Japan. BMC Infect Dis 2016;16(1):650.
- Monavari SHR, Mollaie HR, Fazlalipour M. Simultaneous detection of influenza viruses A, B, and swine origin influenza A using multiplex one-step real-time RT-PCR assay. Appl Biochem and Biotechnol 2014;172(2):984-92.
- Hurt AC. The epidemiology and spread of drug resistant human influenza viruses. Curr Opin Virol 2014;8:22-9.
- Kuo RL, Yang SL, Liu YC, Chen LT, Mok CK, Kuo SM, et al. Influenza A/B virus detection and influenza A virus subtyping with emphasis on the novel H7N9 virus by using multiplex real-time RT-PCR. J Virol Methods 2014;208:41-6.
- Khansarinejad B, Soleimanjahi H, Samiee SM, Hamidieh AA, Paryan M, Sanahmadi Y. Quantitation of human cytomegalovirus DNA in plasma using an affordable in-house qPCR assay. J virol methods 2012;183(2):170-5.
- Sakthivel SK, Whitaker B, Lu X, Oliveira DB, Stockman LJ, Kamili S, et al. Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses. J virol methods 2012;185(2):259-6.
- Sultani M, Azad TM, Eshragian M, Shadab A, Naseri M, Eilami O, et al. Multiplex SYBR Green Real-Time PCR Assay for Detection of Respiratory Viruses. Jundishapur J Microbiol 2015;8(8):e19041.
- Li M, Xie Zhixun, Xie Zhiqin, Liu J, Xie L, Deng X, et al. Simultaneous detection of four different neuraminidase types of avian influenza A H5 viruses by multiplex reverse transcription PCR using a GeXP analyser. Influenza and Other Respir Viruses 2016;10(2):141-9.
- Khandaker G, Zurynski Y, Ridley G, Buttery J, Marshall H, Richmond PC, et al. Clinical epidemiology and predictors of outcome in children hospitalised with influenza A(H1N1)pdm09 in 2009: a prospective national study. Influenza and Other Respir Viruses 2014;8(6):636-45.
- Kidd M. Influenza viruses: update on epidemiology, clinical features, treatment and vaccination. Curr Opin Pulm Med 2014;20(3):242-6.
- Lazarus R, Lim PL. Avian influenza: recent epidemiology, travel-related risk, and management. Curr Infect Dis Rep 2015;17(1):1-9.
- Perez LJ, Perera CL, Coronado L, Rios L, Vega A, Frias MT, et al. Molecular epidemiology study of swine influenza virus revealing a reassorted virus H1N1 in swine farms in Cuba. Prev Vet Med 2015;119(3):172-8.
- Payungporn S, Phakdeewirot P, Chutinimitkul S, Theamboonlers A, Keawcharoen J, Oraveerakul K, et al. Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection. Viral Immunol 2004;17(4):588-93.
- Poddar SK, Espina R, Schnurr DP. Evaluation of a single-step multiplex RT-PCR for influenza virus type and subtype detection in respiratory samples. J Clin Lab Anal 2002;16(3):163-6.
- Qin ZF, Sun J, Lu TK, Zeng SL, Hua QY, Ling QY, et al. Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex). Virol Sin 2012;27(2):120-31.
- Sanghavi SK, Bullotta A, Husain S, Rinaldo CR. Clinical evaluation of multiplex real-time PCR panels for rapid detection of respiratory viral infections. J Med Virol 2012;84(1):162-9.
- Nummi M, Mannonen L, Puolakkainen M. Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens. Springerplus 2015;4(1):1-8.
- Rheem I, Park J, Kim TH, Kim JW, et al. Evaluation of a multiplex real-time PCR assay for the detection of respiratory viruses in clinical specimens. Ann Lab Med 2012;32(6):399-406.
- Detection of influenza virus subtype A by polymerase chain reaction: WHO external quality assessment programme summary analysis, 2015. Wkly Epidemiol Rec 2016;91(1):3-11.
- Haines AM, Tobe SS, Linacre A. Optimization of Diamond Nucleic Acid Dye for quantitative PCR. Biotechniques 2016;61(4):183-9.
- Cheng YH, Kuo CN, Lai CM. Effective Natural PCR-RFLP Primer Design for SNP Genotyping Using Teaching-Learning-Based Optimization with Elite Strategy. IEEE Trans Nanobioscience 2016;PP(99):1-1.
- Liu J, Gratz J, Amour C, Nshama R, Walongo T, Maro A, et al. Optimization of Quantitative PCR Methods for Enteropathogen Detection. PLoS One 2016;11(6):e0158199.
- Lievens A, Jacchia S, Kagkli D, Savini C, Querci M. Measuring Digital PCR Quality: Performance Parameters and Their Optimization. PLoS One 2016;11(5):e0153317.
- Wadle S, Lehnert M, Rubenwolf S, Zengerle R, Von Stetten F. Real-time PCR probe optimization using design of experiments approach. Biomol Detect Quantif 2016;7:1-8.
- Slavov SN, Otaguiri KK, Figueiredo GG, Yamamoto AY, Mussi-Pinhata MM, Kashima S, et al. Development and optimization of a sensitive TaqMan(R) real-time PCR with synthetic homologous extrinsic control for quantitation of Human cytomegalovirus viral load. J Med Virol 2016;88(9):1604-12.
- Jacchia S, Nardini E, Savini C, Petrillo M, Angers-Loustau A, Shim JH, et al. Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay. J Agric Food Chem 2015;63(6):1711-21.
- Lizette Del-Toro-Sanchez C, Villasenor-Alvarado S, Zurita-Martinez F, Castellanos-Hernandez O, Rodriguez-Sahagun A, Isabel Torres-Moran M, et al. Optimization of DNA isolation and PCR protocol for analysis and evaluation of genetic diversity of the medicinal plant, Anemopsis californica using RAPD. Acta Biol Hung 2013;64(2):184-95.
- Camacho JLV, Torres EM, Cadena C, Prieto J, Prieto LV, Torregroza DAV. The use of factorial design, image analysis, and an efficiency calculation for multiplex PCR optimization. J Clin Lab Anal 2013;27(3):249-52.