反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

Evaluation of Reversed Passive Latex Agglutination(RPLA) for The Detection of Clostridium perfringens Type A Enterotoxin from Fecal Specimens

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合，共配成３６４份模拟标本，采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验，Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株），ＲＰＬＡ与生物学试验结果完全一致，均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性，而各生物学试验却均为阴性，该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌，表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８，ＣｐＮＣＴＣ１０６１１，ＣｐＮＣＴＣ１０６１４，ＣｐＮＣＴＣ１０６１２，ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性，但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌，后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６，ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性。

In this paper, we studied the detection of Clostridium perfringens type A (Cp-A) euterotoxin by RPLA and biological assays involving mouse lethallity test, permibility factor test in guinea pigs, and toxicity assay with Vero cell, using a total of 364 imitation samples made by mixing the culture filtrates of 52 strains of toxigenic bacteria and 7 stool mixtures from healthy peoples aged variously (old age, middle age, young, adolescent,children, infant, newborn), respectively.The results demonstrated that all 233 imitation samples made of strains tested,except Cp, were negative for CP-A enterotoxin by RPLA as by every biological test.It means that no false positive result was found in the RPLA assay.Of the 126 imitation samples made of 18 strains of enterotoxigenic Cp-A, 70 were found to be containing enterotoxin which were detected by RPLA,and by biological assays as well.Seven samples made of culture of strain CP NCTC8797 were positive for enterotoxin by RPLA, whereas they were negative when by biological assays. It was confirmed by PCR test that this strain really is enterotoxigenic, indicating the RPLA is more sensitive than biological assays.Fourteen samples made of culture of CP NCTC8678 and CP NCTC8449 respectively,were negative for enterotoxin by both of RPLA and biological assays. These strains seem to have lost their enterotoxigenic genes because PCR also has shown negative.Other 35 samples made of 5 cultures of Cp-A also were negative for enterotoxin by RPLA and biological assays; PCR test showed that they are enterotoxigenic,however. These strains probably didn't produce enterotoxin in the cultures.The conclusion was drown that RPLA is available for the detection of Cp-A enterotoxin in the feces, and may be applied to the screening test at least.