為建立一種鑒定豬急性腹瀉綜合征病毒（SADS-CoV）的RT-PCR方法，根據(jù)NCBI登錄的SADS-CoV N基因保守區(qū)域序列，設(shè)計(jì)了3對(duì)引物（P1、P2和P3），通過(guò)對(duì)SADS-CoV及其他6種病毒進(jìn)行檢測(cè)，篩選出最合適的引物，然后對(duì)退火溫度、引物濃度、dNTPs濃度、rTaq DNA聚合酶濃度和循環(huán)次數(shù)等反應(yīng)條件進(jìn)行了優(yōu)化。結(jié)果顯示，P3引物對(duì)SADS-CoV特異性最好，與其他病毒無(wú)交叉反應(yīng)。敏感性試驗(yàn)顯示，該方法最低檢測(cè)限可達(dá)9.55×102 copies/μL；重復(fù)性試驗(yàn)顯示該方法重復(fù)性良好。運(yùn)用建立的RT-PCR方法，對(duì)廣東省的21份臨床樣品進(jìn)行檢測(cè)，結(jié)果檢出SADS-CoV陽(yáng)性樣品4份，陽(yáng)性樣品的測(cè)序結(jié)果與其RT-PCR完全一致。結(jié)果表明，本研究成功建立了一種鑒定SADS-CoV的RT-PCR方法，且該方法具有檢測(cè)快速、成本廉價(jià)、特異性強(qiáng)、敏感性高和重復(fù)性好的優(yōu)點(diǎn)，可以用于SADS-CoV的臨床診斷。
Establishment and Application of a RT-PCR Assay for Detection of Porcine Acute Diarrhea Syndrome Coronavirus
In order to establish a RT-PCR assay for detection of porcine acute diarrhea syndrome virus（SADS-CoV），three pairs of primers（P1，P2 and P3）were designed based on N gene sequence of SADS-CoV registered in NCBI，and the most appropriate primer was selected through the detection of SADS-CoV and other six kinds of viruses，followed by the optimization of reaction conditions including annealing temperature，primer concentration，dNTPs concentration，rTaq DNA polymerase concentration，cycle index，etc. The results showed that P3 primer was with the best specificity against SADS-CoV，and failed to react with other viruses. It was shown that，by sensitivity test，the lowest detection limit was 9.55×102 copies/μL；the established assay was with good repeatability as tested. 21 clinical samples from Guangdong were detected by the RT-PCR assay，4 positive samples were found，which was consistent with the sequencing results. Therefore，a RT-PCR assay was successfully established for detection of SADS-CoV，which was characterized by rapid detection，low cost，strong specificity，high sensitivity and good repeatability，and could be used for identification of SADS-CoV in practice.
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