基于分枝菌酸的分枝杆菌反相高效液相色谱分型鉴定方法

采用反相高效液相色谱法(HPLC)构建了49种分枝杆菌标准菌株的分枝菌酸指纹图谱库,并对分枝杆菌进行分型鉴定。菌株经培养(对于慢生长分枝杆菌培养3周，快生长分枝杆菌培养1周)后，取两植菌勺的量，皂化1 h后，于4 ℃下储存。通过酸化方法提取分枝菌酸，并用4-溴苯甲酰基溴衍生化,以HPLC分析分枝杆菌衍生物,并以其峰形的分布及峰的相对保留时间、相对峰高为指标对分枝杆菌进行分型鉴定。该法重现性良好,各峰保留时间的相对标准偏差为0.13%～1.07%。根据构建的49种《伯杰细菌鉴定手册》中所载入的分枝杆菌标准菌株的分枝菌酸指纹谱库,发现不同菌种的分枝菌酸的指纹图谱分别具有单簇峰、双簇峰、三簇峰(含多簇峰)的特征。依据相对保留时间和相对峰高的不同,对49种分枝杆菌中的41种进行了分型。结果表明,所建立的反相HPLC法可快速准确地对分枝杆菌进行分型鉴定。

Identification of Mycobacterium species using reversed-phase high performance liquid chromatographic analysis of mycolic acid

A method for the identification of Mycobacterium species using reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The fingerprints library was constructed on the basis of RP-HPLC chromatograms of the mycolic acids derivatives from 49 Mycobacterium species cultures. Two inoculation loops of Mycobacterium, for fast growth with one-week incubation or for slow growth with three weeks incubation, were saponified for 1 h and stocked at 4°C. The mycolic acids from each culture of Mycobacterium species were acidified, extracted, derivatized, and analyzed by the RP-HPLC method. On the basis of the HPLC patterns of relative retention time and relative peak height, the identification of Mycobacterium was performed. This established method has a good precision of retention times with the relative standard deviations (RSDs) ranging from 0.13% to 1.07%. The mycolic acids fingerprints library of HPLC patterns was set up, including 49 species that were recorded in ‘Bergey's Manual of Systematic Bacteriology’. Three patterns were observed from the chromatographic behaviors of mycolic acids derivatives, including single peak-cluster, double peak-clusters, triple and multi peak-clusters. Forty-one species were successfully identified according to the different relative retention times of the peaks and the relative peak heights. The established method can identify Mycobacterium species with rapidity and high reliability.