Comparison of NP and M Primers for Avian Influenza Virus Detection by Reverse Transcription Polymerase Chain Reaction

以NP及M引子逆轉錄聚合酶連鎖反應檢測禽流感病毒的比較

洪惠雯(Hue-Wen Hong) ； 鄭益謙(Ivan-Chen Cheng) ； 陳慧文(Hui-Wen Chen) ； 王金和(Ching-Ho Wang)

《臺灣獸醫學雜誌》 38卷4期 (2012/12) Pp. 213-220

https://doi.org/10.7009/TVJ.201212.0213

摘要

禽流感病毒8段基因中，核蛋白質﹙NP﹚及基質蛋白質﹙M﹚基因較其他基因保守，因此利用此二基因可以檢測所有亞型的禽流感病毒。我們應用已發表的NP引子及M52引子，以逆轉錄聚合酶連鎖反應﹙reverse transcription polymerase chain reaction, RT-PCR﹚及即時逆轉錄聚合酶連鎖反應﹙real-time reverse transcription polymerase chain reaction, rRT-PCR﹚的方法進行禽流感的檢測，為了比較其靈敏度，選取尿囊液的病毒，進行序列稀釋，結果在RT-PCR及rRT-PCR的方法中，M52引子比NP引子皆敏感100倍；以NP引子檢測66個進入雞肉及13個羽毛樣本皆為陰性，以M52引子檢測，有13個雞肉樣本為禽流感陽性，羽毛樣本為陰性；M52引子產物經定序，證實為雞細胞的28SrRNA基因，有偽陽性的出現。為避免雞隻細胞核酸的干擾，以純化的病毒進行RT-PCR，結果M52引子與NP引子的靈敏度相同，結論此NP引子適合禽流感病毒檢測，而此M52引子不適合。

關鍵字

禽流感病毒 ； 靈敏度 ； 偽陽性 ； M52引子 ； Np引子

並列摘要

In detecting all subtypes of avian influenza viruses (AIVs), amplifications of nucleoprotein (NP) or matrix protein (M) genes are better than other genes because they are conserved more among the 8 genome segments in avian influenza virus. We compared the two published primer pairs targeting NP or M genes to detect 66 imported chicken meats and 13 feather samples using reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR (rRT-PCR). Two primer pairs, NP and M52, were used to compare the detection limits for a virus in allantoic fluid. The results showed that the method using the M52 primer pair was 100 times more sensitive than that using the NP primer pair for virus in allantoic fluid. By using these primers in RT-PCR and rRT-PCR, AIV detection results showed that all the samples tested were negative with the NP primer pair. The feather samples were negative with the M52 primer pair as well. However, 13 of 66 chicken meat samples showed positive with the M52 primer pair. The M52 primer pair products were sequenced and proved to be the chicken 28S rRNA gene, considered to be false positive. To avoid the interference of chicken gene, purified AIV without cell debris was used to evaluate these two primer pairs. The results showed that both primer pairs had the same detection limit. Thus, the NP primer pair but not the M52 primer pair is suitable for AIV detection.

並列關鍵字

avian influenza virus ； detection limit ； false positive ； M52 primer ； NP primer