Oakey J, Hawkesford T, Smith C, Hewitson G, Tolosa X, Wright L, Moody N, Rodwell B, Corney B, Waltisbuhl D
Biosecurity Queensland, Queensland Primary Industries and Fisheries, Biosecurity Sciences Laboratory, Yeerongpilly, Queensland 4105, Australia.
Aust Vet J. 2011 Jul;89 Suppl 1:39-42. doi: 10.1111/j.1751-0813.2011.00747.x.
Describe the in-house validation of a previously reported influenza virus type A 5'Taq nuclease assay for detecting equine influenza virus A RNA in nasal swab material.
The validation compares the 5'Taq nuclease assay with a gel-based reverse transcription nested polymerase chain reaction (PCR) previously reported by the Irish Equine Centre for detection of H3N8 and H7N7 equine influenza viruses. This test was chosen because it targets a different region of the viral genome to the real-time test, so it is not merely a repeat of the same test in a different format. Moreover, nested PCRs are commonly considered to have similar sensitivity to real-time PCRs and are therefore ideal for evaluation comparisons.
The sensitivity of the nested PCR was comparable to the 5'Taq nuclease test. Known positive samples and known negative samples reacted with both tests with 100% correlation. Parallel testing of 276 nasal swab samples showed 98% agreement.
The specificity of the nested amplicons was confirmed by nucleotide sequencing and showed >99.5% identity with the same region of previously published equine influenza virus A sequences. The results of this work are appropriate validation for the acceptance of the real-time PCR for equine influenza A virus in equine nasal swabs.
描述一种先前报道的用于检测鼻拭子材料中马A型流感病毒RNA的A型流感病毒5'核酸酶检测法的内部验证情况。
该验证将5'核酸酶检测法与爱尔兰马术中心先前报道的用于检测H3N8和H7N7马流感病毒的基于凝胶的逆转录巢式聚合酶链反应（PCR）进行比较。选择该检测方法是因为它针对病毒基因组的不同区域，而非实时检测法，所以它不仅仅是以不同形式重复相同的检测。此外，巢式PCR通常被认为与实时PCR具有相似的灵敏度，因此非常适合用于评估比较。
巢式PCR的灵敏度与5'核酸酶检测法相当。已知阳性样本和已知阴性样本在两种检测中反应的相关性为100%。对276份鼻拭子样本进行平行检测显示一致性为98%。
通过核苷酸测序确认了巢式扩增子的特异性，并且与先前发表的马A型流感病毒序列的相同区域显示出>99.5%的同一性。这项工作的结果为接受用于马鼻拭子中马A型流感病毒的实时PCR提供了适当的验证。
