Primers for influenza viruses, parainfluenza viruses, adenoviruses, coronaviruses, orthohantaviruses, respiratory syncytial virus and human metapneumovirus, and rhinoviruses.
Several viruses can infect the lower and upper respiratory tract of us humans. The respiratory system allows humans to breathe. The respiratory system includes several organs and structures needed to exchange gases such as oxygen (in) and carbon dioxide (out). Parts of the respiratory system are the nose and nasal cavity, the sinuses, mouth, throat, voice box, windpipe, diaphragm, and the lungs.
Many common viral infections target the upper respiratory system causing severe symptoms in infants, the elderly, and patients with lung or heart problems.
The list of common respiratory viruses includes the epidemic influenza viruses A, B, C, avian influenza viruses, parainfluenza viruses 1–4, adenoviruses, coronaviruses, orthohantaviruses, and respiratory syncytial virus and human metapneumovirus, as well as rhinoviruses.
Symptoms of different respiratory infections, also known as clinical presentation, caused by various viral pathogens, can be very similar. Hence, the correct diagnosis is quite tricky. A rapid virological method will allow a specific and sensitive diagnosis at an early stage of the infection. Significant advances in modern molecular technics have enabled the speedy and sensitive detection of viral pathogens. Polymerase chain reaction (PCR) based methods are now considered as the gold standard of viral assays. For many RNA viruses, including respiratory viruses, multiplex reverse transcription (RT)-PCR assay-based diagnosis allows rapid, sensitive, and specific detection.
Coiras et al., in 2004, developed a multiplex RT-nested PCR assay for the detection and identification of several respiratory viruses.These include the human parainfluenza viruses types 1, 2, 3, and 4AB, the coronaviruses type 229E and OC43, and generic human enteroviruses and rhinoviruses. The researchers designed primers selecting sequences from the conserved regions of haemagglutinin genes, the conserved regions of coronavirus spike protein genes, and the polyprotein gene of rhinoviruses and enteroviruses, between the 5’-non-coding region (5’-NCR) and VP4/VP2 regions. Table 1 lists GenBank accession numbers of the viral sequences, sequences, and properties of all primers studied.
Table 1: Primers for Respiratory Viruses including Human Parainfluenza Viruses (Parainf.), Coronaviruses, Enteroviruses (Enterov.), and Rhinoviruses (Rhinov.) Used in the First Round Multiplex RT-PCR and in the Following Nested PCR (Adapted from Coiras et al.).
Amplification steps and primera Sequence (5’-3’) Gene Gene position Melting temp (°C) G + C content (%) Amplicon size (bp) RT-PCRa 1-PIV13 AGGWTGYSMRGATATAGGRAARTCATA HA Parainf.1 (641-667) Parainf.3 (635-661) 52–60 30–48 Parainf.1 (635) Parainf.3 (635) 2-PIV13 CTWGTATATATRTAGATCTTKTTRCCTAGT HA Parainf.1 (1277-1248) Parainf.3 (1270-1241) 52–56 23–33 1-PIV2 TAATTCCTCTTAAAATTGACAGTATCGA HA Parainf.2 (259-286) 53 29 Parainf.2 (683) Parainf.4AB (1070) 1-PIV4 ATCCAGARRGACGTCACATCAACTCAT 5’NCR-HA Parainf.4 (107-81)c 57–60 41–48 2-PIV24 TRAGRCCMCCATAYAMRGGAAATA HA Parainf.2 (942-919) Parainf.4 (963-940) 49–59 29–54 1-HcoV TGTGCCATAGARGAYWTACTTTTT SP 229E (2068-2090) OC43 (2727-2750) 49–52 29–38 229E (851) OC43 (806) 2-HcoV AACCGCTTKYACCAKCAAYGCACA SP 229E (2919-2896) OC43 (3533-3511) 54–61 42–58 1-EV/RV CTCCGGCCCCTGAATRYGGCTAA 5’NCR-VP4/VP2 Enterov. 445-467d 59–62 57–65 Enterov. (755) Rhinov. (639) 2-EV/RV TCIGGIARYTTCCASYACCAICC 5’NCR-VP4/VP2 Rhinov.1200-1178 53–64 43–68 Nestedb 3-PIV13 ACGACAAYAGGAARTCATGYTCT HA Parainf.1 (754-776) Parainf.3 (748-770) 50–55 35–48 Parainf.1 (439) Parainf.3 (390) 4-PIV1 GACAACAATCTTTGGCCTATCAGATA HA Parainf.1 (1193-1168) 55 38 4-PIV3 GAGTTGACCATCCTYCTRTCTGAAAAC HA Parainf.3 (1138-1112) 57–60 41–48 3-PIV24 CYMAYGGRTGYAYTMGAATWCCATCATT HA Parainf.2 (487-514) Parainf.4 (509-536) 53–63 29–54 Parainf.2 (297) Parainf.4AB (174) 4-PIV2 GCTAGATCAGTTGTGGCATAATCT HA Parainf.2 784-761 54 42 4-PIV4 TGACTATRCTCGACYTTRAAATAAGG HA Parainf.4 683-358 52–56 31–42 3-HcoV TTGTGCGCAATGTTATAAWGGYAT SP 229E (2174–2197) OC43 (2831-2854) 51–52 33–38 229E (630) OC43 (587) 4-HcoV GATAATRTGAGTRCCATTWCCACA SP 229E (2804–2781) OC43 (3418–3696) 51–54 32–42 3-EV/RV ACCRASTACTTTGGGTRWCCGTG 5’NCR-VP4/VP2 Enterov. 536–559c 55–59 48–57 Enterov. (226) Rhinov. (110) 4-EV/RV CTGTGTTGAWACYTGAGCICCCA 5’NCR-VP4/VP2 Rhinov.762–743 55–59 48–57
a1, forward;2,reverse in first-round RT-PCR. b3, forward;4,reverse in nested PCR. cPrimer located up-stream from coding region for haemagglutinin gene. dGene position referred to Poliovirus1strain Sabin (Accession no. V01150). Note: All rhinoviruses have a deletion of approximately 116 bp as regards enteroviruses.
Van de Pol et al. used primers for real-time PCR diagnostic of respiratory viruses from patients admitted with respiratory symptoms. Diagnostic of specific respiratory viruses allows clinicians to initiate optimal patient management and initiate adequate (future) use of antiviral therapy and optimal infection control.
Table 2: Primers and probes for real-time PCR detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses (Adapted from van de Pol et al.).
Virus(es) Target gene Forward primer(s) (5’–3’) Reverse primer(s) (5’–3’) Probe(s)a RSV A Nucleocapsid AGA TCA ACT TCT GTC ATC CAG CAA TTC TGC ACA TCA TAA TTA GGA GTA TCA AT FAM-CAC CAT CCA ACG GAG CAC AGG AGA T-TAMRA RSV B Nucleocapsid AAG ATG CAA ATC ATAAAT TCA CAG GA TGA TAT CCA GCA TCT TTA AGT ATC TTT ATA GTG FAM-TTC CCT TCC TAA CCT GGA CAT AGC ATA TAA CAT ACC T-TAMRA IV A Matrix AAG ACC AAT CCT GTC ACC TCT GA CAA AGC GTC TAC GCT GCA GTC C FAM-TTT GTG TTC ACG CTC ACC GT-TAMRA IV B Hemagglutinin AAA TAC GGT GGA TTA AAC AAA AGC AA CCA GCA ATA GCT CCG AAG AAA FAM-CAC CCA TAT TGG GCA ATT TCC TAT GGC-TAMRA PIV 1 Hemagglutinin-neuraminidase TGA TTT AAA CCC GGT AAT TTC TCA T CCT TGT TCC TGC AGC TAT TAC AGA FAM-ACG ACA ACA GGA AAT C-MGB PIV 2 Hemagglutinin-neuraminidase AGG ACT ATG AAA ACC ATT TAC CTA AGT GA AAG CAA GTC TCA GTT CAG CTA GAT CA FAM-ATC AAT CGC AAA AGC TGT TCA GTC ACT GCT ATA C-TAMRA PIV 3 Hemagglutinin-neuraminidase TGA TGA AAG ATC AGA TTA TGC ATA TC CCG GGA CAC CCA GTT GTG FAM-TGG ACC AGG GAT ATA CTA CAA AGG CAA AAT AAT ATT TCT C-TAMRA PIV 4 Nucleocapsid CAA AYG ATC CAC AGC AAA GAT TC ATG TGG CCT GTA AGG AAA GCA FAM-GTA TCA TCA TCT GCC AAA TCG GCA ATT AAA CA-TAMRA AVs Hexon TTT GAG GTG GAY CCM ATG GA TTT GAG GTY GAY CCC ATG GA AGA ASG GSG TRC GCA GGT A AGA ASG GTG TRC GCA GAT A FAM-ACC ACG TCG AAA ACT TCG AA-MGB FAM-ACC ACG TCG AAA ACT TCA AA-MGB FAM-ACA CCG CGG CGT CA-MGB aFAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binding.
Reference
M.T. Coiras, J.C. Aguilar, M.L. García, I. Casas, and P. Pérez-Breňa; Simultaneous Detection of Fourteen Respiratory Viruses in Clinical Specimens by Two Multiplex Reverse Transcription Nested-PCR Assays. Journal of Medical Virology 72:484–495 (2004). [PMC]
Infectious Diseases
The Respiratory System
Alma C. van de Pol, Anton M. van Loon, Tom F. W. Wolfs, Nicolaas J. G. Jansen, Monique Nijhuis, Els Klein Breteler,1 Rob Schuurman, and John W. A. Rossen; Increased Detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses with Real-Time PCR in Samples from Patients with Respiratory Symptoms. JOURNAL OF CLINICAL MICROBIOLOGY, July 2007, p. 2260–2262. [PMC]
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