Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay
© Ng et al; licensee BioMed Central Ltd. 2006
Received: 16 August 2005
Accepted: 02 March 2006
Published: 02 March 2006
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.
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.
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.
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.
Influenza A virus infects many animals such as humans, pigs, horses, marine mammals, and birds . In avian species, most influenza virus infections cause mild localized infections of the respiratory and intestinal tract, but highly pathogenic strains such as H5N1 cause system infections in which mortality may reach 100% . In humans, influenza viruses cause a highly contagious acute respiratory disease that resulted in epidemic and pandemic disease in humans .
Three types of influenza viruses, types A, B, and C are known and they belong to a family of single-stranded negative-sense enveloped RNA viruses called Orthomyxoviridae . The viral genome is comprised of eight RNA segments (seven in Type C). Influenza A viruses can be classified into subtypes based on antigenic differences in the two surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) which are required for viral attachment and cellular release. Other major viral proteins include the nucleoprotein (NP) which is the main structural protein, membrane proteins (M1 and M2), polymerase proteins (PA, PB1 and PB2), and non-structural proteins (NS1 and NS2). Currently, sixteen subtypes of HA (H1-H16) and nine NA (N1-N9) antigenic variants are known in influenza A virus mostly related with veterinary significance, with only three subtypes circulating in humans (H1N1, H1N2, and H3N2). However, in recent years, the pathogenic H5N1 subtype of avian influenza A has been reported to cross the species barrier and infect humans as documented in Hong Kong in 1997 and 2003 [5–7]. Since late 2003, the H5N1 avian A influenza in poultry reached epidemic proportions with reports of serious outbreaks in several Asian countries including Vietnam, Thailand, South Korea, Laos, Cambodia, Indonesia, Japan and Malaysia [8, 9] that resulted in massive culling of millions of poultry which had severe economic repercussions.
As a result, H5N1 avian influenza A virus represents a potential danger to human health not only in Asia but to the world. Therefore, in addition to containment procedures, sensitive detection assays for early diagnosis are vital to lower the chances of spread and reduce the risk of development into an epidemic. Current methods employed to detect H5N1 subtypes include various polymerase chain reaction (PCR) assays [10–12, 7] and antigen tests using various fluorescence and enzyme-linked immunoassays . However, these assays are reported to be low in specificity and sensitivity, and clinically, the low sensitivity of these diagnostics may limit the usefulness for reliable detection of influenza A (H5N1) virus in humans . Therefore, there is an urgent need for improved, validated, sensitive diagnostic tests for rapid and early diagnosis. In this study, we describe the development of a nucleic acid detection test that is rapid, specific and sensitive, thus allowing greatly improved detection of the H5N1 avian influenza A virus.
Extraction of total RNA was performed following manufacturers' protocol from QIAamp Viral RNA Mini Kit (Qiagen, Germany) and TRIZOL (Invitrogen, USA) using all necessary safety precautions. The resultant RNA was dissolved in 20 μl of RNase-free water.
2 μl of RNA was used in 25 μl reaction mixtures using the One-Step reverse transcription (RT)-PCR system (Qiagen, Germany) with H5N1 specific primers (forward primer: 5'-ACTATGAAGAATTGAAACACCT-3' and reverse primer: 5'-GCAATGAAATTTCCATTACTCTC-3'). The PCR program was set as: 60°C for 1 min, 42°C for 10 min, 50°C for 30 min, and 94°C for 15 min followed by 35 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min and lastly followed by 72°C for 10 min. The size of this PCR product was 456 bp and was resolved in 1.2 % agarose gels. PCR products were sequenced directly to confirm the identity of the products.
Results and Discussion
Establishment of a new H5N1 PCR primer set
Specific detection of H5N1 avian influenza A
Human specimens used as controls in one-step RT-PCR H5N1 assay
Early disease symptoms
Respiratory syncytial virus (RSV) B
Severe respiratory syndrome virus (SARS)
High fever, dyspnea, malaise
Hepatitis B virus (HBV)
Fever, malaise, dyspnea
Detection of avian influenza A H5N1 subtype from field specimens.
Positive by H5N1 viral isolation
Positive by RT-PCR H5N1 primers
Cloacal and Tracheal swab
Homogenized Pooled tissue and organs
Vietnam and Malaysia
Sensitivity of H5N1 primers compared to WHO recommended H5 primers.
Limit of detection WHO recommended H5 primers
Limit of detection H5N1 primers described here
In conclusion, we have reported an efficient, specific and sensitive assay that has been evaluated on field specimens to be able to detect a wide variety of H5N1 influenza virus isolates. Accurate and sensitive detection of viral RNA is also strongly influenced by the sample type. The rapid one-step single tube reaction described here not only reduce the detection time but also lowers the risk of cross-contamination which has a higher probability in two-steps RT-PCR methods. This cost effective gel-based system has a lower limit of detection in the picogram range which is equivalent to 1 × 103 copies, and is designed to cater for use in the field in regions where real-time PCR platform and equipments may not be available. Clearly, the results would be further strengthened with the inclusion of more known H5N1 influenza in archived samples from humans, but the availability of human samples are difficult due to the low number of human infections at this point. However, observations from this study strongly suggest that the primers are specific for H5N1 which can be very useful for the early detection and monitoring of avian influenza outbreaks.
We thank Jer-Ming Chia, Martin L. Hibberd, Christopher W. Wong and Jian-Jun Liu (Genome Institute of Singapore), No-Na Yeoh, Geok-Huai Ong and Rafidah Ahmad Johari (Veterinary Research Institute, Malaysia), Fook-Kheong Ng, Chee-Wee Lim and Wai-Kwan Wong (Central Veterinary Laboratory, Agri-Food and Veterinary Authority of Singapore), and Kian-Leong Ong and Woon-Hsi Chin (Veredus Laboratories Singapore) for their contributions.
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