腹泻患儿粪便标本中腺病毒PCR检测方法的建立

Developing of a method for detection and identification of entero-adenovirus in fecal specimens from infantile diarrhea

目的建立一种从患腹泻婴幼儿粪便标本中腺病毒的方法。方法设计出可同时鉴定腺病毒并判断是否属于F组（肠道腺病毒Ad40或Ad41）的2组引物，通过PCR检测50份腹泻患儿粪便标本，对鉴定为阳性标本的扩增产物进行测序，以验证腺病毒型别。同时用抗原检测的方法鉴定粪便标本中的腺病毒，并以此为金标准，评价PCR方法检测的敏感度、特异度、阳性预测值、阴性预测值和准确性。结果腺病毒阳性的粪便标本通过PCR扩增可得到大小为301bp的片段，如果属F组型别则还可同时扩增得到1条541bp（Ad40）或586bp（Ad41）的片段。PCR检测的50份粪便标本中有15份为腺病毒阳性，阳性率为30％其中8份属腺病毒F组。15份腺病毒阳性标本的PCR扩增产物经测序比对均属腺病毒，其中13份为F型别，2份为腺病毒7型。以测序结果为金标准，PCR检测F组腺病毒方法的特异度为100％，敏感度为61．6％，阳性预测值为100％，阴性预测值为71．4％，准确性为67．0％。与腺病毒抗原检测法（阳性率为22％）比较，PCR检测腺病毒的敏感度为45．5％，特异度为74．4％，阳性预测值为33．3％，阴性预测值为82．9％，准确性为68．0％。结论成功建立PCR法可一次快速检出患儿粪便标本中的腺病毒，并区分F组和非F组型别，有望用于开展常规粪便标本腺病毒检测，并为肠道腺病毒流行病学的研究提供参考。

Objective To establish a method for detecting and identificating of adenovirus from stool samples from diarrheal infants. Methods Two pairs of primers were designed by the published sequences of adenovirus, one for amplifying adenovirus for all serogroups and the other for amplifying the adenoviruses in group F （ Ad 40/41 ） specifically. Fifty fecal specimens collected from hospitalized infants with diarrhea were tested for adenovirus by the PCR developed in this study addition to adenovirus antigen detection. The PCR products were sequenced and the sequences were compared with the results from PCR. Results The products by amplifying with PCR from adenovirus type 7 and fecal specimens with adenovirus other than group F were 301 bp in length, whereas those from adenovirus group F in fecal samples were 2 bands 301 bp and 541/586 bp as expected. Fifteen out of 50 stool samples tested were positive for adenovirus by PCR （ positive rate 30% ） and all of these were proved to be adenovirus by sequencing analysis. Eight out of 15 adenovirus positive samples were F subgroup by PCR, while 13 were proved to be F subgroup by sequencing. This PCR method showed 100% specificity, 61.6% sensitivity, 100% positive predictive value, 71.4% negative predictive value and 67.0% total coincidence rate for detecting F subgroup adenovirus. Comparing with adenovirus antigen detecting kit, the PCR method for detection of adenovirus displayed 74. 4% specificity, 45.5% sensitivity, 33.3% positive predictive value, 82. 9% negative predictive value and 68. 0% total coincidence rate. Conclusion The PCR developed in this study is found to be a rapid and reliable method for the detection of adenoviruses in infant diarrheal stools samples.