摘要
目的 利用显色法芯片技术建立快速鉴定分枝杆菌菌种的方法.方法 根据美国国家生物技术信息中心(NCBI)提供的分枝杆菌16S rDNA基因的保守区和突变区(第129～267位核苷酸的A突变区、第430～500位核苷酸的B突变区)分别设计引物和寡核苷酸探针,用地高辛标记引物并制备玻璃芯片.采用双重PCR技术分别对16种分枝杆菌标准株、5种非分枝杆菌标准株和120株分枝杆菌临床分离株(非结核分枝杆菌40株、结核分枝杆菌复合群80株)进行扩增,扩增产物分别与玻璃芯片进行杂交检测,尼龙膜显色,以显现蓝黑色斑点作为阳性信号,并根据其在芯片方阵中的位置判断分枝杆菌种类.根据芯片杂交结果,选取部分经显色法芯片技术检测的临床分离株进行DNA测序.结果 16种分枝杆菌标准株和120株分枝杆菌临床分离株经PCR扩增均各产生2条DNA片段,其中1条长度为272～280 bp,1条长度为183～192 bp.16种分枝杆菌标准株均与芯片上特异性探针杂交,应用显色法芯片技术分析16种分枝杆菌标准株和5种非分枝杆菌标准株的特异性为100%.120株分枝杆菌临床分离株均与分枝杆菌属探针a杂交,其中79株确定为结核分枝杆菌复合群,38株确定为非结核分枝杆菌(不产色分枝杆菌17株,胞内分枝杆菌8株,猿猴分支杆菌6株,瘰疬分枝杆菌5株,偶然分支杆菌2株),另3株只与分枝杆菌属探针a杂交,没有鉴定到种.选取的26株分枝杆菌临床分离株(结核分枝杆菌复合群8株,不产色分枝杆菌5株,胞内分枝杆菌、猿猴分枝杆菌、未鉴定到种的分枝杆菌各3株,瘰疬分枝杆菌、偶然分枝杆菌各2株)DNA测序显示,未鉴定到种的3株分枝杆菌中,1株为结核分枝杆菌突变株,1株为 Mycobacterium lentiflavum,1株为 Mycobacterium arupense,芯片上无后2种菌株的特异性探针;其余23株菌株测序结果与芯片检测结果一致.结论 显色法芯片技术能简便、快速、灵敏、特
Objective To establish a method for rapid identification of mycobacterium by using genes chips technique. Methods An primer and oligo-nucleotides probe was designed according to the same sequences along the both sides of the mutation section in the mycobacterium 16S rDNA gene sequence, and the 129th-267th nucleotide acid mutation section A, the 430th-500th nucleotide mutation section B, which were provided by the National Center for Biotechnology Information (NCBI) in theUSA. The primer was labeled with digoxin, and then glass chips were prepared. Afterwards, 16 mycobacterial reference strains, 5 non-mycobacterial reference strains, and 120 mycobacterial clinical isolates (including 40 non-tuberculosis mycobacterial strains and 80 tuberculosis mycobacterial complex strains) were amplified by double PCR technique. The hybridization between the amplified products and the gene chips was examined, and presented using nylon membrane. Dark blue dots were considered positive signals. The type of the mycobacteria was identified based on the position of the dark blue dots in the chips phalanx. According to the hybridization results, some of the clinical isolated strains were selected and sequenced by using gene chips technique. Results After being amplified, both the 16 mycobacterial reference strains and 120 mycobacterial clinical isolates produced two DNA segments (sized 272-280 bp and 183-192 bp, respectively). By using the gene chips technique, a specificity of 100% was achieved in both the 16 mycobacterial and the 5 non-mycobacterial reference strains. All the 16 mycobacterial reference strains showed positive hybridization with the special probe in chips, and the 120 mycobacterial isolates showed positive hybridization with the mycobacterium genus probe a. Among the mycobacterial isolates, 79 strains were identified as tuberculosis mycobacterial complexes, 38 as non-tuberculosis mycobacteria (17 as mycobacterium non-chromogenicum, 8 mycobacterium intracellulare, 6 mycobacterium simiae, 5 mycobacterium scrofulaceum,
出处
《中国医药生物技术》
CSCD
2007年第6期422-427,共6页
Chinese Medicinal Biotechnology
基金
上海市科委重点专项基金(06DZ22320)
关键词
芯片分析技术
分枝杆菌属
DNA
细菌
聚合酶链反应
Microchip analytical procedures
Mycobacterium
DNA, bacterial
Polymerase chain reaction
