Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay

Abstract

Background: Continuous outbreaks of the highly pathogenic H5N1 avian influenza A in Asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. We report here the development of a PCR-based assay that is highly specific for the H5N1 avian influenza A virus.

Methods: A one-step reverse-transcription PCR assay was developed to detect the H5N1 avian influenza A virus. The specificity of the assay was shown by testing sub-types of influenza A virus and other viral and bacterial pathogens; and on field samples.

Results: Validation on 145 field specimens from Vietnam and Malaysia showed that the assay was specific without cross reactivity to a number of other infuenza strains as well as human respiratory related pathogens. Detection was 100% from allantoic fluid in H5N1 positive samples, suggesting it to be a reliable sampling source for accurate detection.

Conclusion: The assay developed from this study indicates that the primers are specific for the H5N1 influenza virus. As shown by the field tested results, this assay would be highly useful as a diagnostic tool to help identify and control influenza epidemics.

Figure 1

Detection of H5N1 avian influenza A virus by one-step RT-PCR. A. Amplification of serially diluted in vitro-transcribed single-stranded RNA (lanes 2 to 11) measured by RiboGreen RNA quantitation reagent and H5N1 RNA extracted from allantoic fluid of infected egg (lane 19). The non-template control is (sterile water) illustrated as "NTC". The viral load is indicated by the number of copies per reaction, (lane 2) 1 × 10⁹ copies per reaction, (lane 3) 1 × 10⁸ copies per reaction, (lane 4) 1 × 10⁷ copies per reaction, (lane 5) 1 × 10⁶ copies per reaction, (lane 6) 1 × 10⁵ copies per reaction, (lane 7) 1 × 10⁴ copies per reaction, (lane 8) 1 × 10³ copies per reaction, (lane 9) 1 × 10² copies per reaction, (lane 10) 10 copies per reaction, and (lane11) 1 copy per reaction. Viral RNA extracted from human specimens that were previously confirmed respiratory syncytial virus (RSV) B (lane 12), dengue virus 1 (lane 13), dengue virus 2 (lane 14), dengue virus 3 (lane 15), dengue virus 4 (lane 16) and severe acute respiratory syndrome (SARS) (lane 17) were also tested as known negatives. RNA extracted from healthy individual (lane 18) was also performed. B. Specific detection of H5N1 avian influenza A virus. Reference strains of different subtypes of avian influenza A viruses (lanes 20 to 24), including non-influenza viruses such as Newcastle disease virus (NDV, lane 25) are indicated. The H5N1, H3N8, H9N2 and NDV isolates were isolated from field samples by the Veterinary Research Institute, Malaysia, the H5N3 and H7N5 isolates were provided by the Department of Veterinary Pathology of Tottori University, Japan. Negative signals from non-H5N1 isolates and the non-template control (water) are shown.

Figure 2

Specific detection of H5N1 avian influenza A virus from archived human and avian samples. A. A panel of archived avian and human subtypes (H1 to H16) of Influenza A virus was tested against the H5N1 primers. Viral RNA were extracted from H1 (lane 5, A/Duck/Victoria/23/81); H2 (lane 6, A/Singapore/1/57); H3 (lane 7, A/Sydney/5/97); H4 (lane 8, A/Stint/Australia/1/2004); H5 (lane 9, A/Avian/L2640C); H6 (lane 10, A/Avian/WA/2727/78); H7 (lane 11, A/Chicken/Victoria/85); H8 (lane 12, A/Turkey/Ontario/6118/67); H9 (lane 13, A/Chicken/Malacca/4905/2003); H10 (lane 14, A/Chicken/Germany/n/49); H11 (lane 15, A/Sandpiper/Australia/6/2004); H12 (lane 16, A/Stint/WA/574/84); H14 (lane 17, A/Mallard/Gurjev/244/82); H15 (lane 18, A/Avian/WA/1762/78); H16 (lane 19, A/Gull/Denmark/68110/62). Two human archived RNA samples, Human 279 (lane 3, A/Hong Kong/279/2003) and Human 3028 (lane 4, A/Vietnam/3028/2004) were tested positive. Positive H5N1 RNA sample from in vitro-transcribed single-stranded RNA was used (lane 2), and the non-template control (water) is shown. B. The same set of RNA samples were tested against the matrix gene. The 978 bp fragment was detected in all samples (lanes 22 to 36) as shown.

Figure 3

Sensitivity test against human and avian strains. A. The H5N1 primers were tested for sensitivity against the currently recommended WHO H5 primer set using three H5N1 strains, one human strain from stored archive RNA, and two freshly extracted avian strains. Human 3028 (A/Vietnam/3028/2004, lanes 3 to 11); Avian 933 (lA/Chicken/Vietnam/933/2004, lanes 15 to 23), and Avian 949B (A/Chicken/Vietnam/949B/2004, lanes 24 to 32) are shown. Serial dilution was performed on the RNA samples from 10^-1 to 10^-8 as indicated. The 219 bp fragment amplified using the WHO H5 primer set was also performed in parallel as indicated. B. Sensitive detection of H5N1 avian influenza A virus. Five H5N1 samples from Vietnam that have been confirmed by viral isolation and RT-PCR were tested in parallel with an existing H5 primer set (lanes 67 to 71) and the H5N1 primer set (lanes 73 to 77) described in this report. Only three out of five samples were detected by the H5 primer set (lanes 67 to 69), while the new H5N1 primer set detected all five samples (lanes 73 to 77).