应用DNA微阵列技术快速鉴定分枝杆菌菌种

Rapid identification of Mycobacterium species by DNA chip

目的利用DNA微阵列的高通量、高效性,建立一种快速、简便的分枝杆菌分子菌种鉴定方法,为临床医师正确诊断提供依据。方法以DNA直接测序法为对照,通过PCR-SSCP和DNA微阵列技术分析28种分枝杆菌标准菌株、9种非分枝杆菌和465株分枝杆菌临床分离株的菌种。结果应用DNA微阵列技术分析28种分枝杆菌标准菌株和9种非分枝杆菌菌株,特异性100％。465株分枝杆菌临床分离株中,经16S rRNA PCR-SSCP初步菌种鉴定,256株为结核分枝杆菌复合群,应用DNA微阵列分析,显示与分枝杆菌属探针M和结核分枝杆菌复合群探针a杂交阳性,两种鉴定方法结果一致;209株PCR-SSCP初步鉴定为非结核分枝杆菌的分离株,经芯片分析,68株为龟分枝杆菌龟亚种和脓肿亚种,46株为胞内分枝杆菌,34株为堪萨斯、瘰疬、胃和猿猴分枝杆菌复合群,31株为偶然分枝杆菌,16株为戈登分枝杆菌,3株为鸟分枝杆菌,2株为海和溃疡分枝杆菌复合群,1株为土分枝杆菌, 1株为迪氏分枝杆菌,1株为草分枝杆菌;另6株只与探针M杂交,经测序显示5株为胞内分枝杆菌,但其基因序列与标准菌株不完全相同,1株为新金色分枝杆菌,芯片上无鉴定该菌种的探针。结论用DNA微阵列可简便、快速、灵敏、特异地将大多数分枝杆菌鉴定到种,提高分枝杆菌病的正确诊断率,指导临床合理治疗。

Objective To utilize the high-scale and high efficiency of DNA chip to develop a rapid, simple, and specific method for identification of Mycobacterium species, to provide a basis for the correct diagnosis of mycobacterium diseases. Methods DNA sequencing was used as the control, the target DNA fragments of 28 Mycobacterium reference strains, 9 non-Mycobacterium strains and 465 Mycobacterium isolates were analyzed by PCR-SSCP and DNA chip. Results The reference strains of 28 Mycobacteria and 9 non-Mycobacteria were analyzed by DNA chips, the results showed that the DNA chip was specific. Of 465 mycobacterium Clinical isolates, 256 strains were identified as Mycobacterium tuberculosis complex （MTBC）with PCR-SSCP, and were positive hybridization with Mycobacterium genus probe M and MTBC-specific probe a on DNA chip. 209 strains were identified as non-tuberculosis mycobacteria with PCR-SSCP, and their species were identified by DNA chips as follows： 68 were M. chelonae , 46 were M. intracellulare , 34 were M. kansasii , M. gastri , M. scrofulaceum , M. simiae complex,31 were M.fortuitum, 16 were M. gordonae,3 were M. avium,2 were M. marinum and M. abscess complex, 1 was M. terrae, lwas M. diernhoferi, and 1 was M. phlei .6 strains that were only positive hybridization with probe M were sequenced and showed that 5 were M. intracellulare, which had different DNA sequences with the reference strain, and 1 was M. neoaurum. There is not specific probe for M. neoaurum on DNA chip.Conclusion It might be a simple, rapid, sensitive and specific method for identifieation of most Mycobacterium species byDNA chip, and it could raise correct diagnosis rate of Mycobacterium diseases, and direct the physicians toperform rational chemotherapy.