Establishment and Application of Fluorescent Quantitative RT-PCR for Detection of H3 Subtype Avian Influenza Virus
The avian influenza virus(AIV)of subtype H3 infects a wide range of hosts and is capable of cross-species transmission.In addition to causing disease in poultry,it also causes many cases of human infection,which is of great public health importance.Therefore,it is necessary to establish a rapid and sensitive method for the detection of H3 subtype AIV.In this study,the H3 subtype AIV HA gene sequence published by GenBank in recent years was used to design a pair of specific primers and TaqMan probes in the conserved region.By optimising the reaction conditions and drawing the standard curve using cRNA standard and viral nucleic acid standard as templates,a fluorescence quantitative RT-PCR detection method for H3 subtype AIV was established and its specificity,sensitivity and repeatability were evaluated.The method was further validated using laboratory-challenged chicken tissue samples and clinical swab samples.The results showed that the method was highly specific and did not cross-react with other subtypes of AIV and common avian pathogens.The lower detection limits of the method were 1.0×102 copies·μL-1 and 102 EID50·0.1 mL-1,respectively,and the sensitivity was 10-fold higher than that of conventional RT-PCR.The coefficient of variation within and between groups was less than 1.5％and the repeatability was good.The results of the animal challenge clinical test and clinical swab samples showed that the sensitivity of the method was higher than that of the conventional RT-PCR method,and the concordance rate was 100％,which could be used for clinical detection.In conclusion,the fluorescent quantitative RT-PCR detection method for H3 subtype AIV established in this study has the characteristics of specific,rapid and sensitive,which provides certain technical support for the rapid diagnosis,monitoring,prevention and control of H3 subtype AIV.
万方数据
维普
