显色法芯片技术快速鉴定分枝杆菌菌种

 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、特异地将大多数分枝杆菌鉴定到种，具有一定的临床推广应用价值。

Rapid identification of mycobacterium by genes chips technique

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, and 2 mycobacterium fortuitum), and 3 only showed positive hybridization with mycobacterium genus probe a, and thus could not be identified. DNA sequence showed that among the 3 strains, which could not be identified, one was mycobacterium tuberculosis mutant, one was mycobacterium lentiflavum, and the other was mycobacterium arupense. In the genes chips, no specific probe for the latter two mycobacterial strains was detected. The remaining 23 strains showed the same sequencing results as those shown by the chips test. Conclusion Genes chips technique is a convenient, rapid, sensitive, and specific method for the identification of mycobacteria with great practical value.