结核病分子流行病学在结核病的控制方面有着良好的应用前景,发达国家已将其列入常规检查,在我国也有很好的应用前景和更大的应用空间.目前的基因分型的主流方法仍然是采用数目可变串联重复序列(VNTR)和间隔区寡核苷酸分型共同应用的模式.间隔区寡核苷酸分型是继IS6110限制性片段长度多态性(RFLP)之后应用于结核分枝杆菌基因分型最广泛的方法之一,目前被作为鉴定北京基因型菌株的标准方法.VNTR方法操作简便、快速,成本低廉,重复性好,便于不同实验室间相互比较；VNTR方法选用的位点在不同国家和地区会有不同,VNTR位点分辨率的高低也需要深入研究.IS6110-RFLP方法虽然已经不再大规模地使用,但是一直作为鉴定其他基因分型方法的正确率和准确度的金标准.随着分子生物学技术的进步,基因分型技术出现新的发展趋势.如长片段多态性(LSP)和单核苷酸多态性(SNP)方法是近几年出现的频率越来越高的分型方法.目前LSP和SNP方法的分辨率虽然不高,但在研究系统发育中确有很好的应用.系统发育学的研究一开始是采用VNTR-15和VNTR-24的方法,后来发现存在不确定性且误差较大,随着对LSP和SNP特性的了解更加全面,认为LSP和SNP是研究系统发育学的最好的方法.随着分子生物学技术的不断进步,结核病分子流行病学的研究将会为结核病的预防和控制提供更多支持和指导.

Molecular epidemiology of tuberculosis has a good prospect in tuberculosis control,and developed countries have applied it in the routine examination.In our country,there will be much greater application prospects and space.The current genotyping mainstream mode is common application of variable number tandem repears (VNTR) and spoligotyping.Spoligotyping is one of the most widely used methods applied to Mycobacterium tuberculosis genotyping after IS6110-restriction fragment length polymorphism (RFLP) method occurred and is used as a standard method for identification of Beijing genotype strains.VNTR method is simple,fast,low cost,repeatable,and easily compared between different laboratories.The VNTR loci selected in different countries and regions are different,different VNTR locus has different discriminatory index and the level needs further study.IS6110 RFLP method is no longer widely used,but it has been regarded as a gold standard for the identification of the accuracy of other g

目的：利用加入突变TaqDNA聚合酶等的优化方法实现人类牙齿龈沟液中的细菌及人类基因组片段的直接扩增。方法随机选取10例牙龈炎患者。用无菌注射器分别吸取10μL的龈沟液。然后各吸取2μL，标记为第一组，分别加入突变TaqDNA聚合酶及DMSO，通过改变PCR反应体系，直接实现龈沟液中细菌的16sRNA及MAOA-VNTR基因的扩增；再分别吸取2μL，标记为第二组，此组先采用微量DNA提取试剂盒来实现DNA的提取，然后扩增龈沟液中细菌的16sRNA及MAOA-VNTR基因。结果直接扩增法所获得的扩增产物比传统提取DNA 法多2个，两种方法的转化子阳性率无差别；MAOA-VNTR可扩增出单倍体，串联体等。结论直接PCR法可以用于龈沟液中细菌的初步检测以及人类基因组小片段的扩增，也可应用于牙龈炎或牙周炎患者的口腔龈沟液研究及临床诊断。

Objective To achieve the direct amplification of bacteria in gingival cervical fluid ( GCF) and fragments of the human genome by using the optimization method of joining mutant TaqDNA polymerase .Methods Ten cases of patients with gingivitis were selected randomly , and 10μL of GCF from them were absorbed by a sterile syringe respectively .The samples were divided into two groups , the first group was added with mutant TaqDNA polymerase and DMSO in 2 μL GCF respectively , the 16 sRNA and MAOA-VNTR gene of bacteria were amplifed di-rectly by changing the reaction system of PCR .The second group , DNA was extracted using DNA extraction kit firstly and then amplified the 16 sRNA and MAOA-VNTR gene of bacteria in 2 μL GCF.Results Two more amplification products were obtained by direct amplification rather than traditional method .There was no significant difference between the two methods on transformants positive rate; Haploidy and concatemer were amplified by MAOA -VNTR.Con-

多位点可变数目串联重复序列分析(MLVA)是以可变数目串联重复序列(VNTR)为基础的一种流行病学基因分型方法.由于该分型方法具有分辨率高、重复性好、结果数字化便于室间对比等特点,已被广泛应用于各种细菌的分子流行病学研究中.此文就该分型技术的基本原理、优越性及其在沙眼衣原体的分型和分子流行病学研究中的应用进行综述.

Multilocus variable-number of tandem-repeats analysis (MLVA) is an epidemiological genotyping method based on the variable-number tandem-repeats (VNTR).It has been widely used in molecular epidemiological research of various bacteria because of its high resolution,good reproducibility and digital results which are convenient for comparison among different laboratories.In this review,the basic principles and advantages of MLVA,and its application in genotyping and molecular epidemiological research of Chlamydia trachomatis are summarized.

目的初步评价15个可变串联重复序列(VNTR)位点对结核分枝杆菌临床分离株的分型能力,寻找适合湖州地区结核分枝杆菌基因分型的位点。方法 2011年1—4月连续收集湖州地区结核分枝杆菌临床分离株,采用多位点数目多位点串联系复序列(MLVA)分析方法,对15个VNTR位点进行基因多态性检测,分析单个位点以及15个位点组合的基因分型能力(Hunter-gaston指数,HGDI)。结果分辨率较好的多态性位点有2个:Mtub-21和Mtub-39;分辨率中等的多态性位点共7个:ETR-A,ETR-C,MIRU-10,MIRU-23,MIRU-27,MIRU-40和Mtub-30;分辨率较差的多态性位点有6个:ETR-B,ETR-D,ETR-E,MIRU-16和MIRU-26和MIRU-39。通过计算,本次研究运用15个位点进行基因分型的HGDI为0.99,分辨能力好。结论 VNTR位点对结核分枝杆菌基因的分辨力较好,但还需继续寻找其他丰富多态性的VNTR位点,以便快速分型,提高分辨力。

Objective To preliminarily evaluate the ability of 15 -loci variable number tandem repeats on the genotyping of clinical isolates of mycobacterium tuberculosis,and explore the suitable loci for genotyping of Mycobacterium tuberculosis in Huzhou City.Methods From 201 1 January to 201 1 April,the clinical isolates of mycobacterium tuberculosis were selected from Huzhou area to test the polymorphism of every loci by using MLVA detection method and analyze hunter-gaston discriminatory index of individual locus as well as combinations of 15 locus.Results There were two locus of Mtub-21 and Mtub-39 which had better degree of polymorphism;seven locus of ETR-A,ETR-C,MIRU-10,MIRU-23,MIRU-27,MIRU-40 and Mtub-30 had moderate degree of polymorphism;six locus of ETR-B,ETR-D,ETR-E,MIRU-16,MIRU-26 and MIRU-39 had poor degree of polymorphism.By calculation,the result showed that HGDI was 0.99 by using 15 locus to genotype,which indicated the ability to distinguish the genotype was good.Conclusion We w

目的：探讨IL-1β、IL-1ra血清水平及白细胞介素-1B（ IL-1B）和白细胞介素-1RN（ IL-1RN）基因多态性与HCV相关肝病发生发展的关系。方法以酶联免疫吸附试验（ ELISA）测定IL-1β、IL-1ra血清水平；基因芯片技术检测310例HCV肝病和324例不相关联的健康对照人群的IL-1B-31 C/T、IL-1B-511C/T单核苷酸多态性（SNP）；琼脂糖凝胶电泳检测IL-1RN内含子2（intron 2）基因多态性（ VNTR）。聚合酶链反应-限制性片段长度多态性（ PCR-RFLP）随机分析部分样本验证基因芯片分型结果；HCV基因分型采用直接测序法；Lightcycler 特异性荧光探针PCR检测HCV RNA载量；血清丙氨酸氨基转移酶（ alanine aminotransferase ，ALT）使用ROCHE cobas 8000全自动生化分析仪检测。结果（1）病例组IL-1β血清水平（22.6±7.3） pg/ml显著高于对照组（13.7±4.2） pg/ml（ P＜0.01）；IL-1ra血清水平（286.30±55.10） pg/ml显著高于对照组（185.55±48.32） pg/ml（P＜0.01）。（2）在病例组、轻中型肝炎、重型肝炎、肝硬化合并肝癌组、1b型组及2a型组，IL-1B-511T等位基因携带者血清IL-1β水平均显著高于其相应CC纯和子及对照组（ P＜0.05），且HCV感染各组-511 T携带者IL-1ra/IL-1β比值显著低于对照组（P＜0.05）；IL-1B-511T携带者在1b型组的IL-1β水平较2a型组显著增高（P＜0.05）；IL-1B-511T携带者IL-1ra血清水平显著高于相应对照组（P＜0.05）；IL-1B-511CC携带者血清IL-1β水平在各组别间差异无统计学意义（P＞0.05）。（3）病例组IL-1B-511TT基因型频率显著高于对照组（ P＜0.05，OR＝1.55，95％CI＝1.10～2.18），两组间T等位基因频率差异有统计学意义（P＜0.05，OR＝1.31，95％CI＝1.05～1.63）。 IL-1RN VNTR二组间差异无统计学意义（P＞0.05）。（4）IL-1B-511C/T SNP基因型频率在不同转归类型中差异有统计学意义（ P＜0.005）。与相应的CT

Objective To study the characteristics of IL-1B-31/-511 single nucleotide polymor-phisms (SNPs) and the variable number tandem repeat (VNTR) in intron 2 of the IL-1ra gene (IL-1RN) in patients with HCV-related liver diseases .Methods The concentration of IL-1βand IL-1ra in serum sam-ples was measured by ELISA assay .The SNPs of IL-1B gene (-31C/T,-511C/T) from 310 cases with HCV infection and 324 unrelated healthy controls were determined by using gene chip analysis , and the results for some randomly selected specimens were compared with those by using polymerase chain reaction -restriction fragment length polymorphism ( PCR-RFLP) assay.The VNTR polymorphism of IL-1RN intron 2 was ana-lyzed by PCR-RFLP assay.The serum level of alanine aminotransferase (ALT), an indicator of hepatocellu-lar injury, was detected by ROCHE cobas 8000 analyzer.HCV replication was measured by using specific fluorescence PCR .The genotypes of HCV were determined by direct nucleotide sequencing test .Results