绵羊梨型虫PCR方法的建立及序列分析

Establishment of PCR method and sequence analysis of sheep Ggiardia

本研究建立了绵羊梨型虫的检测方法,并对退火温度和循环次数进行梯度优化。选取条带最亮且无杂带的温度和循环次数作为PCR检测方法的优化结果,将新建的PCR方法与传统的吉姆萨染色法结果进行对比,将阳性的PCR产物进行测序。结果表明:PCR反应的最佳体系为94℃预变性5 min;94℃变性1 min,56℃退火90s,72℃延伸1min,共循环39次;72℃延伸10min,4℃保存;在50份临床样品的检测中,PCR法检出的阳性样品有13份,阳性率为26%(13/50),吉姆萨染色发检出的阳性样品有8份,阳性率为16%(8/50),由PCR方法检测出的阳性样品与吉姆萨染色发检出的阳性样品符合率达100%。阳性PCR产物的测序结果的同源性分别为100%和98%。本研究证实PCR法的灵敏度和准确性高于吉姆萨氏染色镜检法。

The study established a test method for sheep Giardia. The test conditions, such as an- nealing temperature and cycle times were optimized by choosing the most bright band. The new method was compared with Giemsa staining. The result showed that the best system of PCR reac- tion is predegeneration at 94℃ 5 min,degeneration at 94℃ 1 rain,annealing at 56℃ 90 s,extention 1 min,a totle of thirty-nine round, extention at 72℃ 10 min,preservation at 4℃. Among the fifty clinical samples, 13 positive samples were tested by PCR,the positive rate is 26 % （13/50）, 8 posi- tive samples were tested by Giemsa staining, the positive rate is 16 % （8/50）. Positive samples tex- ted by PCR was the same as Giemsa staining. The sequencing homology was 100% and 98%. The sensitivity and accuracy of PCR method were higher than those of Giemsa staining.