套式聚合酶链反应检测伤寒杆菌DNA及其临床应用

DETECTION OF SALMONELLA TYPHI DNA BY NESTED POLYMERASE CHAIN REACTION AND ITS CLINCAL APPLICATION

应用套式聚合酶链反应（ｎｅｓｔｅｄ－ＰＣＲ）技术，建立了扩增伤寒杆菌鞭毛蛋白基因多变区特异核苷酸序列的方法。对９株沙门氏菌及７株其他临床分离菌进行检测，结果仅伤寒杆菌扩增出３４８ｂｐ特异性片段，以γ－３２ｐＡＴＰ标记的为伤寒杆菌特异的寡核苷酸探针对扩增产物做ｓｏｕｔｈｅｒｎ印迹杂交，证实上述结果可靠。扩增灵敏度为３０ｆｇ伤寒杆菌ＤＮＡ（约１０个菌体）。对６９例发热待查患者的７８份血标本进行检测，其敏感性（１００％）明显高于传统（血，骨髓）培养法（７３％），Ｐ＜０．０１；特异性均为１００％。另外，其检出敏感性不受预先抗菌治疗的影响，整个实验在１０～ｌ２小时内完成。以上表明ｎｅｓｔｅｄ－ＰＣＲ是一种快速、敏感、特异诊断伤寒的方法。

he nested polymerase chain reaction(nested-PCR)has been adopted to amplifythe speeifie sequences of the hypervariable region of the flagellin gene of SalmonellaTyphi(S. typhi).9 strains of Salmonella(including two strains of S. typhi) and 7strains of non salmonella organisms were tested. The results showed that only S.typhi amplified products had 343bp specific DNA f ragment. That amplified productswere analysed by Southern blot hybridization using γ32-PATP labelled 40 base probespeeifie for S. typhi the above results were proved reliable. The sensitivity of amp-lifieation was about 30fg DNA of S. typhi (corresponding to 10 organisms). It wasused to detect S. typhi, DNA in 78 blood specimens colleected from 69 patients withtyphoid fever, it was shown that the sensitivity of nested-PCR(100%)was signifi-cantly higher than that of traditional(blood and/or bone marrow) culture method(73%,p<0.01).The specificity were both l00%. Sensitivity of nested-PCR was notaffected by prior antibiotic treatment. The whole procedure was completed within l0-12h. The above results suggest that nested-PCR may be a specific,sensititive andrapid technique for the diagnosis of typhiod fever.