分枝杆菌菌种鉴定DNA微阵列的初步研究与应用

Rapid identification of mycobacterial species by DNA microarray

目的制备快速鉴定分枝杆菌菌种的DNA微阵列。方法根据19种分枝杆菌标准株的16SRNA序列设计寡核苷酸探针,制备DNA微阵列,通过反向杂交技术鉴定19种分枝杆菌标准株、9种非分枝杆菌标准株和31株分枝杆菌临床分离株带荧光标记的PCR产物。结果该DNA微阵列与9种非分枝杆菌标准株不杂交,具有分枝杆菌特异性;可将19种分枝杆菌标准株鉴定到群或种,具有较高的分枝杆菌种特异性。应用PCR-SSCP分枝杆菌初步菌种鉴定方法对31株分枝杆菌临床分离株进行初步的鉴定,9株为结核分枝杆菌复合群和22株为非结核分枝杆菌。应用DNA微阵列进一步进行菌种鉴定,9株与探针a杂交阳性,为结核分枝杆菌复合群;2株与探针c杂交阳性,为胞内分枝杆菌;8株与探针d杂交阳性,为堪萨斯分枝杆菌或瘰疬分枝杆菌或猿猴分枝杆菌或胃分枝杆菌;1株与探针k杂交阳性,为戈登分枝杆菌;2株与探针p杂交阳性,为龟分枝杆菌脓肿亚种;2株与探针r杂交阳性,为偶然分枝杆菌;1株与探针s杂交阳性,为草分枝杆菌;2株与探针a和p杂交阳性,为结核分枝杆菌和龟分枝杆菌脓肿亚种复合感染株;1株与探针a和r杂交阳性,为结核分枝杆菌和偶然分枝杆菌复合感染株;3株与该芯片杂交阴性。2株临床分离株经基因测序也证实DNA微阵列鉴定的特异性。结论该DNA微阵列有可能简便、快速。

Objective To prepare a DNA microarry for the identification of mycobacterial species. Method According to the 16S RNA sequences from 19 mycobacteria standard strains, oligonucleotide probes were designed and DNA microarrays were prepared. Nine non-mycobacterial and 19 mycobacterial standard strains, 31 M.tuberculosis clinical isolates were amplified by PCR, and then identified by DNA microarrys. Results The DNA microarrays did not hybridized with 16S rDNA fragments from 9 non-mycobacteria standard strains, which is specific for mycobacteria. The microarrays could specifically identified 19 mycobacteria to groups or species. Thirty-one mycobacterial clinical isolates were identified as 9 M.tuberculosis complex and 22 non-~tuberculous mycobacteria by PCR-SSCP. Using the microarrays, 16S rDNA PCR products from 9 isolates ~hybridized with probe a on microarrys and were identified as M.tuberculosis complex; 2 isolates hybridized with probe c and were identified as M.intracellulare; 8 isolates hybridized with probe d and were identified as ~M.kansasii -scrofulaceum-gastri-simiae group; 1 isolates hybridized with probe k and were identified as M.gordonae; 2 isolates hybridized with probe p and were identified as M.abscessus; 2 isolates hybridized with probe r and were identified as M.fortuitum; 1 isolates hybridized with probe s and were identified as M.phlei; 2 isolates hybridized with probe a, p and were identified as M.tuberculosis complex and M.abscessus; 1 isolates ~hybridized with probe a and r were identified as M.tuberculosis complex and M.fortuitum; 3 isolates did not ~hybridize with the microarrys. 16S rDNAs from 2 isolates of them were sequenced, and the results were consistent with that from the microarrys. Conclusion The most mycobacterial species might be identified simply, rapidly and correctly by DNA microarrys.