Multiplex PCR for Typing and Subtyping Influenza and Respiratory Syncytial Viruses

Abstract

A multiplex reverse transcription (RT)-PCR method that has been developed is capable of detecting and subtyping influenza A (H1N1 and H3N2) and B viruses as well as respiratory syncytial virus (RSV) types A and B in respiratory clinical samples taken as part of a national community-based surveillance program of influenza-like illness in England and Wales. The detection of each different pathogen depended on distinguishing five amplification products of different sizes on agarose gels following RT-PCR with multiple primer sets. The multiplex RT-PCR was tested with 65 nasopharyngeal apirates from which RSV had been isolated and 237 combined nose and throat swabs from which influenza A (H1N1 and H3N2) or B virus had been detected by virus isolation, as well as 40 respiratory samples from which other viruses including cytomegalovirus, herpes simplex virus, enteroviruses, and parainfluenza viruses had been grown. For the typing and subtyping of influenza A and B viruses and RSV types A and B, the multiplex RT-PCR gave an excellent (100%) correlation with the results of conventional typing and subtyping with specific antisera. Multiplex RT-PCR can also be used to accurately detect more than one viral template in the same reaction mixture, allowing viral coinfections to be identified with the same respiratory specimen.

Comparison of four nasal sampling methods for the detection of viral pathogens by RT-PCR—A GA2LEN project

“…PCR for RV was done with 6 l cDNA and OL26 and OL27 primers (Papadopoulos et al, 2000). PCR for Influenza virus (serotypes AH1, AH3, B) was done in two rounds (nested-PCR); in the first round mixture 4 l of cDNA were added and 2 l of primary product were then transferred to 48 l of the secondary amplification mixture using a second primer set internal to that of the first round (Stockton et al, 1998). PCR for Adenovirus was done with 4 l of cDNA in a single round, according to Freymuth et al (1997).…”

Viruses detected by systematic multiplex polymerase chain reaction in adults with suspected community-acquired pneumonia attending emergency departments in France

“…Since the advent of PCR, the advances in these new molecular techniques have shown their applicability to improve the detection of agents causing various types of infections. The multiplex PCR method allows the potential amplification of many nucleic acid targets within a single reaction and has proven to be an attractive diagnostic method for multi-pathogen detection in respiratory tract infections [9,10].…”

Semiquantitative one-step RT-PCR for simultaneous identification of human influenza and respiratory syncytial viruses

“…Acute respiratory tract infections (ARIs) are the most common causes of morbidity and mortality in young children, the elderly and immune-compromised patients worldwide (Boivin et al, 2004;Hinman, 1998). Among the major viral causative agents of ARI are influenza A and B viruses and the respiratory syncytial viruses (RSVs) A and B (Stockton et al, 1998). Rapid diagnosis of these viral pathogens is important for determining appropriate clinical intervention and for monitoring these or related viruses during outbreaks or pandemic of emerging pathogens.…”

“…In the past few years, several multiplex reverse transcription-polymerase chain reaction (mRT-PCR) methods have been developed for the detection of influenza viruses, RSV and parainfluenza viruses (PIVs), etc. (Bellau-Pujol et al, 2005;Coiras et al, 2003;Grondahl et al, 1999;Stockton et al, 1998;Syrmis et al, 2004). In these methods, analysis of PCR products relies either on agarose gel electrophoresis or more time-consuming enzyme-linked assays and many of them include a nested PCR step to enhance sensitivity.…”

“…In addition, the optimal of multiplicity for real-time PCR is currently limited to three due to a limited number of fluorophores (S. Cheng, unpublished). The method reported in this study, which primarily targets influenza A/H3, A/H1 and B viruses and RSV A and B, is carried out in a singlestep reaction with minimal opportunity for contamination since no secondary or tertiary nested PCR is needed as in previous methods (Bellau-Pujol et al, 2005;Coiras et al, 2003;Grondahl et al, 1999;Stockton et al, 1998). The PCR amplicons in this method range from 133 to 390 bp, which are relatively small in size that can increase the assay sensitivities.…”