几种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法的验证

目的比较和验证5种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法。方法首先使用美国NCBI网站的Primer—BLAST程序验证5种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法引物的理论特异性，然后使用猪流感H1N1病毒核酸、2009年新甲型H1N1流感病毒核酸、H5N1禽流感核酸、能力验证样品作为试验样品验证方法的实验特异性。结果理论验证结果显示5对来源于文献的引物均适合于猪流感的检测，前3对可用于新型甲型H1Nl流感的检测，但只限于个别来源的毒株，与预期略有差别。实验验证结果显示，方法l可正确检出甲型流感病毒；方法2可正确检出猪流感H1N1亚型病毒、新甲型H1N1流感病毒核酸；方法3检测新甲型H1N1流感核酸时未获得阳性结果；方法4可以正确检测出猪流感H1病毒核酸；方法5可以正确检测出猪流感H1N1病毒核酸，但检测新甲型H1N1流感核酸、禽流感H5N1核酸时也为阳性。结论方法1可用于猪甲型流感和新甲型H1N1流感病毒的初筛；方法2可用于猪流感H1N1亚型病毒和新甲型H1N1流感病毒核酸的初筛；方法3不适合新甲型H1N1流感病毒核酸的检测；方法4可用于猪流感H1亚型的检测；方法5不适合于猪流感H1N1亚型病毒的检测。采用SYBRGreenI荧光RT—PCR方法检测甲型H1N1流感病毒核酸，特异性普遍不十分理想，只能用于初筛检测。

Validation of SYBR Green I Fluorescent RT - PCR methods for detecting several H1N1 influenza A Virus

Abstract Objective To compare and validate 5 SYBR Green I fluorescent RT - PCR methods for the detection of nucleic acid from H1 N1 influenza A virus. Methods 5 methods are theoretically validated by using Primer - BLAST program on NCBI website, and experimentally validated by detecting nucleic acid from swine influenza A virus H1N1, 2009 novel H1N1 influenza A virus, avian influenza A virus HSN1 and 6 samples from CNAS profi- ciency test. Results By theoretical validation, all of the 5 SYBR Green I fluorescent RT - PCR methods demon- strate suitability for the detection of swine influenza A virus H1N1, methods 1 - 3 can be used for the detection of 2009 novel H1 N1 influenza A virus, but limite within individual strain, which is of a slight differences with the ex- pected. In experimental validation, method 1 achieves the anticipated results as detecting all kinds of influenza A virus; Method 2 shows positive results while detecting swine influenza A virus H1 N1 and 2009 novel H1 N1 influen- za A virus; Method 3 does not display positive while detecting 2009 novel H1 N1 influenza A virus; Method 4 can detect swine influenza A virus H1 ; Method 5 is able to detect swine influenza A virus H1N1, and also present posi- tive results when detecting 2009 novel H1N1 influenza A virus and avian influenza A virus HSN1. Conclusion A- mong 5 SYBR Green I fluorescent RT - PCR methods, method 1 can be used in the screen detection of nucleic acid from swine influenza A virus and novel H1 N1 influenza A virus, method 2 is proper for screen detection of swine in- fluenza A virus H1 N1 and 2009 novel H1 N1 influenza A virus, method 3 is not suitable to detect novel H1 N1 influ- enza A virus, method 4 can be used for detection of nucleic acid from swine influenza A virus H1, method 5 is not suitable for the detection of swine influenza A virus H1N1. When detecting influenza A virus H1N1, SYBR Green I fluorescent RT- PCR methods can be only used in screen detection, since their specificities are not obvious.