Multiplex RT-PCR For Rapid Detection Of Common Respiratory Virus

Respiratory viruses are a series of pathogens,which can evoke respiratory infection diseases,and have been increasingly shown to play a role in the morbidity and mortality of elder or immunosuppressed patients. Besides, they are highly contagious in the populations and can easily cause world-wild pandemic. Because it's very difficult to identify the specific kind of respiratory virus by clinical symptom or radiological indication, the test of pathogenic microorganisms is extremely important in disease diagnoses and supervise. However,traditional diagnostic methods have limitation in test sensitivity and feasibility in many contexts, and focus only on partial viral pathogens. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory viruses. As is well known, seasonal influenza A virus (INF A),influenza B virus (INF B),Human rhinovirus (HRV) and Swine-Origin H1N1 Influenza Virus (SOIVs)are four important respiratory RNA virus which are hard to detect, we tried to develop a multiplex RT-PCR method to identify these virus rapidly and accurately.Objective: To develop a simultaneous and rapid test technique for important viral pathogens which may cause respiratory infection.Methods: A total of 36 nasopharyngeal swabs from upper respiratory tract infection Patients were collected in Xinqiao Hospital from September 2009 to October 2009. The viral RNA were extracted and tested by traditional RT-PCR method. Plasmids pBS-TⅡ,which include genome sequence of INF A,INF B, HRV and SOIVs, were served as standard preparation for detection. Under optimized PCR conditions, a multiplex RT-PCR technique was developed to simultaneously detect four virus and have been Compare with traditional RT-PCR by detecting the same clinical specimens from upper respiratory tract infection Patients.Resμlts: There were 12 positive results in 36 specimens, including 3 INF A, 1 INF B, 3 HRV and 5 SOIVs. The percentage of SOIVs in total influenza virus was 55.6%. Four specific RT-PCR products, 212bp for INF A, 450bp for INF B, 389bp for HRV and 153bp for SOIVs, were amplified respectively by multiplex RT-PCR. The LOD of INF A, INF B, HRV and SOIVs were 104,104,103,104copies/ml. The specificity of the technique was verified by detecting another 6 common pathogens including Streptococcus pneumonia, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, escherichia coli and enterococcus faecalis. None of these were found positive in the same multiplex PCR conditions. Compare to traditional RT-PCR, multiplex PCR method has shown similar effect.Conclusions:SOIVs are the predominant strains of influenza virus in Chongqing in 2009 autumn.The multiplex RT-PCR assay may be used as a rapid, sensitive and specific diagnostic method for the differentiation of INF A, INF B, HRV and SOIVs.