Żaczek A, Brzostek A, Kuroń A, Wojtasik A, Sajduda A, Dziadek J
Department of Biochemistry and Cell Biology, University of Rzeszów, Rzeszów, Poland.
Institute of Medical Biology, Polish Academy of Science, Łódź, Poland.
Int J Tuberc Lung Dis. 2014 Mar;18(3):302-9. doi: 10.5588/ijtld.13.0538.
There is a need for rapid, inexpensive methods for analysing a limited number of Mycobacterium tuberculosis strains. The ligation-mediated polymerase chain reaction (LM-PCR) method appears to be sufficiently discriminative and reproducible to be considered as a molecular tool for the initial evaluation of hospital outbreaks, laboratory cross-contamination, and family or small community transmission.
To develop a new LM-PCR method based on PCR amplification of the 5'-flanking region of insertion sequence (IS) 6110 consisting of SalI/PvuII digestion of chromosomal DNA, ligation of a SalI linker and differentiation of IS6110-carrying restriction fragments by suppression subtractive hybridisation.
The fast ligation amplification polymorphism (FLAP) method was applied in the analysis of 62 M. tuberculosis clinical isolates and compared with IS6110-restriction fragment length polymorphism (RFLP) and mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) analyses of the same strains.
The sensitivity of FLAP was estimated at 0.25 ng/l. FLAP yielded 32 patterns among the 62 M. tuberculosis strains compared to respectively 28 and 36 patterns obtained using MIRU-VNTR and IS6110-RFLP. Its Hunter-Gaston discriminatory index value (0.973) is similar to that of MIRU-VNTR (0.966) and IS6110-RFLP (0.971). The specificity of the FLAP patterns was also confirmed.
FLAP proved highly discriminating, sensitive and specific and could be a valuable molecular tool, especially for analysing a limited number of M. tuberculosis strains.
需要快速、廉价的方法来分析有限数量的结核分枝杆菌菌株。连接介导的聚合酶链反应（LM-PCR）方法似乎具有足够的鉴别力和可重复性，可被视为用于医院暴发、实验室交叉污染以及家庭或小社区传播的初步评估的分子工具。
基于对插入序列（IS）6110的5'侧翼区域进行PCR扩增来开发一种新的LM-PCR方法，该方法包括染色体DNA的SalI/PvuII消化、SalI接头的连接以及通过抑制性消减杂交对携带IS6110的限制性片段进行区分。
将快速连接扩增多态性（FLAP）方法应用于62株结核分枝杆菌临床分离株的分析，并与同一菌株的IS6110限制性片段长度多态性（RFLP）和分枝杆菌散布重复单位可变数目串联重复序列（MIRU-VNTR）分析进行比较。
FLAP的灵敏度估计为0.25 ng/μl。在62株结核分枝杆菌菌株中，FLAP产生了32种图谱，相比之下，使用MIRU-VNTR和IS6110-RFLP分别获得了28种和36种图谱。其亨特-加斯顿鉴别指数值（0.973）与MIRU-VNTR（0.966）和IS6110-RFLP（0.971）相似。FLAP图谱的特异性也得到了证实。
FLAP被证明具有高度鉴别力、灵敏度和特异性，可能是一种有价值的分子工具，尤其适用于分析有限数量的结核分枝杆菌菌株。
