Relevance of MUC1 mucin variable number of tandem repeats polymorphism in H pylori adhesion to gastric epithelial cells

AIM:To evaluate the influence of MUC1 mucin variable number of tandem repeats (VNTR) variability on H pylori adhesion to gastric cells. METHODS: Enzyme linked immunosorbent assay (ELISA)-based adhesion assays were performed to measure the adhesion of different H pylori strains (HP26695 and HPTx30a) to gastric carcinoma cell lines (GP202 and MKN45) and GP202 clones expressing recombinant MUC1 with different VNTR lengths. RESULTS: Evaluation of adhesion results shows that H pylori pathogenic strain HP26695 has a significantly higher (P < 0.05) adhesion to all the cell lines and clones tested, when compared to the non-pathogenic strain HPTx30a. Bacteria showed a significantly higher (P < 0.05) adhesion to the GP202 cell line, when compared to the MKN45 cell line. Furthermore, both strains showed a significantly higher (P < 0.05) adhesion to GP202 clones with larger MUC1 VNTR domains. CONCLUSION: This work shows that MUC1 mucin variability conditions H pylori binding to gastric cells. The extent of bacterial adhesion depends on the size of theMUC1 VNTR domain. The adhesion is further dependent on bacterial pathogenicity and the gastric cell line. MUC1 mucin variability may contribute to determine H pylori colonization of the gastric mucosa.

Associations between IL-1RN variable number of tandem repeat, IL-1β (-511) and IL-1β (+3954) gene polymorphisms and urolithiasis in Uighur children of China

Objective:Interleukin-1(IL-1)is a pro-inflammatory cytokine which may be related to urolithiasis.Genetic polymorphisms of the interleukin-1beta(IL-1β)have been proposed as markers for urolithiasis in some areas.Due to the high incidence of urolithiasis in Uighur children(Xinjiang,China)and existence of ethnic difference,our aim is to explore the potential of IL-1 gene polymorphisms and urolithiasis among these children.Methods:Genomic DNA extracted from peripheral blood of 115 patients and 98 controls were used for genotype polymorphisms analyses.IL-1 receptor antagonist(IL-1RN)gene variable number of tandem repeat(VNTR)gene polymorphisms were analyzed by PCR method.PCR-based restriction analysis was done for the IL-1β(-511)and IL-1β(+3954)gene polymorphisms by endonucleases Ava I and Taq I,respectively.The genotype distribution,allele frequencies,carriage rate,and haplotype frequencies were statistically analyzed.Results:No significant differences were observed in genotypic frequencies between pediatric urolithiasis patients and control group for IL-1RN gene(χ^(2)=1.906,p=0.605),IL-1β(-511)gene(χ^(2)=0.105,p=0.949),or IL-1β(+3954)gene(χ^(2)=3.635,p=0.169).There were yet no significant differences of the allele frequencies of IL-1RN VNTR gene(p=0.779),IL-1β(-511)gene(p=0.941),and IL-1β(+3954)gene(p=0.418)in the case and control groups,as well as the carriage rate and haplotype of them(all p>0.05).Conclusions:The associations between IL-1RN VNTR,IL-1β(-511)and IL-1β(+3954)genes polymorphisms and urolithiasis were not significant in Uighur children.The results need to be confirmed in studies with larger population sample size,as well as in other ethnic groups.

云南省昭通地区结核分枝杆菌临床分离株遗传多样性和耐药分子特征

目的调查云南省昭通地区结核分枝杆菌(Mycobacterium tuberculosis,MTB)临床分离株的遗传多样性和耐药分子特征。方法2022年9月—2023年8月从昆明医科大学附属昭通医院收治的298例涂阳肺结核患者中共采集了MTB 298株,用MeltPro TB测定法通过荧光聚合酶链反应(polymerase chain reaction,PCR)熔解曲线法鉴定所有分离株。用多色熔解曲线分析技术进行间隔区寡核苷酸分型(interval oligonucleotide typing,Spoligotyping)。用分枝杆菌散布重复单元(mycobcterial interspersed repetitive units,MIRU)-可变数目串联重复序列分型(variable number of tandem repeat typing,VNTR)(MIRU-VNTR)24位点分型法进行基因分型。使用PCR和靶向测序检测耐药基因突变。结果北京菌株占所有MTB菌株的69.80%(208/298)。其他谱系包括T、LAM、MANU2、CAS、NEW-1和SITVITWEB数据库中未发现的孤儿型,分别占5.70%(17/298)、3.36%(10/298)、0.67%(2/298)、0.34%(1/298)、8.39%(25/298)及11.74%(35/298)。北京菌株对左氧氟沙星的耐药率略高于非北京菌株(P=0.042),而北京菌株与非北京菌株2种基因型菌株对异烟肼、利福平、乙胺丁醇、链霉素、吡嗪酰胺、卡那霉素耐药率差异无统计学意义(P>0.05)。MIRU-VNTR 24位点分型分析结果显示,33个菌株可分为13株聚类,其聚类率为39.39%。每个等位基因的Hunter-Gaston指数在0~0.814之间,其中18个位点对非北京菌株具有中高鉴别能力。但只有10个位点对北京菌株具有中高鉴别能力。结论MTB菌株在中国云南省昭通地区表现出较高的遗传多样性,北京菌株是MTB和耐药结核病菌株的优势谱系。

Genetic Testing of the mucin I gene-Variable Number Tandem Repeat Single Cytosine Insertion Mutation in a Chinese Family with Medullary Cystic Kidney Disease

Background： Medullary cystic kidney disease （MCKD） is clinically indistinguishable from several other autosomal-dominant renal diseases： thus, molecular genetic testing is needed to establish a definitive diagnosis. A specific type of single cytosine insertion in the variable number tandem repeat （VNTR） of the mucin 1 （MUC1） gene is the only known cause of MCKD1; however, genetic analysis of this mutation is difficult and not yet offered routinely. To identify the causative mutation/s and establish a definitive diagnosis in a Chinese family with chronic kidney disease, clinical assessments and genetic analysis were performed, including using a modified genotyping method to identify the MUC1-VNTR single cytosine insertion. Methods： Clinical data from three patients in a Chinese family with chronic kidney disease were collected and evaluated. Linkage analysis was used to map the causative locus. Mutation analysis of uromodulin （UMOD） gene was performed using polymerase chain reaction （PCR） and direct sequencing. For MUC1 genotyping, the mutant repeat units were enriched by Mwol restriction, and then were amplified and introduced into pMD-18T vectors. The 192 clones per transformant were picked up and tested by colony PCR and second round of Mwol digestion. Finally, Sanger sequencing was used to confirm the MUC1 mutation. Results： Clinical findings and laboratory results were consistent with a tubulointerstitial lesion. Linkage analysis indicated that the family was compatible with the MCKDI locus. No mutations were found in UMOD gene. Using the modified MUC1 genotyping method, we detected the MUC1-VNTR single cytosine insertion events in three patients of the family; and mutation-containing clones were 12/192, 14/192, and 5/96, respectively, in the three patients. Conclusions： Clinical and genetic findings could support the MCKDI diagnosis. The modified strategy has been demonstrated to be a practical way to detect MUCI mutation.

猕猴桃溃疡病病原菌MLVA分型引物的筛选及验证

【目的】筛选出一组可精准快速地对丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv.actinidiae,Psa)进行分型的引物组合。【方法】针对前期文献已报道的34对引物,采用PCR技术验证该34对引物对中国Psa菌株的扩增效率及准确性;利用模拟PCR获取菌株串联重复(TR)数;以辛普森指数(Simpson’s index,SI)作为筛选引物组合的标准,基于R软件平台,筛选最优引物组合。【结果】34对引物对中国Psa扩增效果均良好,其中TR14与TR11II、TR19与Psa-01引物序列相同;TR8与Psa-08、TR39II与Psa-10、GM-1834与TR10I、GM-1553与TR64II、TR19Psa-01与TR19II扩增同一TR;Psa-09扩增产物串联重复单元长度不唯一,TR2II扩增产物侧翼变异较大,不能通过电泳确定串联重复数;最终确定SI值与全部引物组合相同的最低引物数量为9对,使用该9对引物的组合可将Psa已知的5种生物型准确分开。【结论】TR23/Psa-04、Psa-03、Psa-05、Psa-06、TR10IGM-1834、TR30I、TR1II、Psa-10TR39II、TR64IIGM-1553等9对引物可代表当前文献报道的34对引物,进行Psa分型研究,探索猕猴桃溃疡病的传播和流行规律,为病害防控策略的制定提供科学依据。

抽动秽语综合征汉族患者的执行功能及与多巴胺D4受体第3外显子48bp可重复序列多态性

目的:了解多巴胺D4受体基因第3外显子48bp可重复序列多态性(DRD4 exonIII NTR)与抽动秽语综合征(Gillesdela Tourette syndrome,GTS)患者执行功能缺陷之间的关系。方法:对86例GTS患者进行威斯康星卡片测验(Modified Wisconsin Card sortingtest,WCST)、Stroop色词测验(Strooptest)和连线测验,并和51例正常对照组进行比较;利用PCR技术对GTS患者进行了DRD4exonIII48bpVNTR分析。结果:与正常对照组比较,GTS组在Stroop测验中的C错误数[(44.39±65.3)vs.(20.50±10.85),P<0.01]等(包括C纠错数、C时间、CW正确数、CW错误数、CW纠错数和CW时间)、连线测验A时间[(69.80±25.84)vs.(35.69±8.25),P<0.01](包括连线B时间、错误数、犯规数)、WCST正确数[(44.39±65.37)vs.(27.49±10.85),P<0.01](错误数、持续错误数、非持续错误数和分类数)等测验项目上成绩较差,从共病情况来看,注意缺陷多动综合征共病组在Stroop测验部分项目上比单纯GTS组要差;从GTS组内分析来看,DRD4exonIII48bpVNTR和各神经心理学测验成绩没有关联。结论:抽动秽语综合征患者存在执行功能缺陷,DRD4exonIII48bpVNTR和抽动秽语综合征执行功能缺陷之间可能没有关联。

Genetic Diversity and Drug Susceptibility of Mycobacterium tuberculosis Isolates in a Remote Mountain Area of China

Objective We determined the genetic diversity of Mycobacterium tuberculosis（MTB） in a remote mountainous area of southwest China and evaluated the resolving ability of single nucleotide polymorphism（SNP） genotyping combined with variable number tandem repeat（VNTR） genotyping for Beijing family strains in association with drug resistance status.Methods Three hundred thirty-one MTB strains were isolated from patients living in mountainous regions of southwest China,and 8-loci SNP,VNTR-15 genotyping assays,and drug susceptibility testing of 9 drugs were performed.Results A total of 183 [55.29%（183/331）] strains were classified into the Beijing family.Of the 183 strains,111（60.66%） were defined as modern Beijing strains.The most predominant modern Beijing sub-lineage and ancient Beijing sub-lineage were Bmyc10 [39.34%（72/183）] and Bmyc25 [20.77%（38/183）],respectively.Of the isolates,19.64%（65/331） were resistant to at least 1 of the 9 anti-TB drugs and 17 [4.98%（17/331）] MTB isolates were multi-drug resistant tuberculosis（MDR-TB）.Two hundred sixty-one isolates showed a clustering rate of 14.18%（37/261） and a discriminatory index of 0.9990.The Beijing lineage exhibited a significantly higher prevalence of MDR-TB,as well as resistance to isoniazid（INH）,rifampin（RIF）,and para-aminosalicylic acid（PAS） when analyzed independently（P = 0.005,P = 0.017,P = 0.014,and P = 0.006 respectively）.The Beijing lineage was not associated with genetic clustering or resistance to any drug.In addition,genetic clustering was not associated with drug resistance.Conclusion MTB strains demonstrate high genetic diversity in remote mountainous areas of southwest China.Beijing strains,especially modern Beijing strains,are predominant in remote mountainous area of China.The combination of 8-loci SNPs and VNTR-15 genotyping is a useful tool to study the molecular epidemiology of MTB strains in this area.

Association of serotonin transporter gene polymorphisms and major depressive disorder in Chinese Han population

BACKGROUND： Serotonin transporter （5-HTT） polymorphisms comprise 5-HTT gene-linked polymorphism region （5-HTTLPR） and variable number of tandem repeats （VNTR）. Studies have revealed an association between 5-HTT polymorphism and major depressive disorder, which suggests that the ＂S＂ allele of 5-HTTLPR and Stin2.9 of 5-HTTVNTR are associated with major depressive disorder. However, there are a number of studies that do not support the 5-HTT polymorphism effect in major depressive disorder. OBJECTIVE： To study the relationship between 5-HTT gene polymorphism and major depressive disorder in Chinese Han population. DESIGN, TIME AND SETTING： Case-controlled study of 5-HTT gene polymorphism. The experiment was performed at the Central Laboratory, Third Affiliated Hospital of Sun Yat-sen University, China from March 2005 to January 2006. PARTICIPANTS： A total of 99 depressive patients of Chinese Han nationality were recruited for this study. All patients met DSM-IV diagnostic criteria for major depressive disorder and had a total score of Hamilton Depression Scale （24 items） ≥21 points. In addition, 101 healthy subjects, matched for age and gender, served as the control group. METHODS： Venous blood was collected from all subjects. 5-HTT genotypes and alleles were determined by polymerase chain reaction. Consistent with the Hardy-Weinberg equilibrium, the association between 5-HTT gene polymorphism and major depressive disorder were analyzed by Chi-square test. MAIN OUTCOME MEASURES： 5-HTTLPR and 5-HTTVNTR genotypes and allele frequencies were measured. RESULTS： No significant differences in 5-HTTLPR genotypes and allele frequencies were determined between patients and controls （P 〉 0.05）. However, significant differences in 5-HTTVNTR genotypes and allele frequencies were detected （P 〈 0.01 ）. The Stin2.10 allele and 10/10 genotype associated with major depressive disorder （OR = 2.61,7.7, P 〈 0.05; analysis of dose-response relationships Х^2 = 12.35, P 〈 0.01）. CONCLUSION： Results from the present study revealed no association between 5-HTTLPR and major depressive disorder. However, a significant association between 5-HTTVNTR and major depressive disorder existed in a population of Chinese Han. The presence of Stin2.10 and 10/10 genotypes increased the risk for major depressive disorder in a dose-dependent manner.

Study on genetic polymorphisms of DCC gene VNTR in esophageal cancer

Objective: To observe the distribution pattern of genetic polymorphisms of gene variable number tandem repeat (VNTR) in the deleted gene in colorectal cancer (DCC) on Shaanxi people, and discuss the possible association between it and the susceptibility of esophageal cancer. Methods: Polymorphisms of DCC gene VNTR was studied with PCR in blood samples of 56 unrelated individuals and 49 esophageal cancer samples and 34 pericancerous samples in Shaanxi people. Results: There were 11 alleies wing from 167 bp to 210 bp in all subjects. The range of allele frequencies was from 0.009 to 0. 188, and allele A3, A4, A7 and A9 were more frequent in the control. The polymorphism information content(PIC) of DCC gene VNTR was 0.879,the heterozygosity(H) was 73.2% in the control. The allele frequency of DCC gene VNTR polymorphism in esophageal cancer was significantly different from that of control(P < 0. 01 ). Conclusion: Our findings suggest that polymorphism of DCC gene VNTR might be associated with the susceptibility of esophageal cancer.

Significance of IL-1Ra and IL-6 gene variants in Turkish patients with Crimean-Congo hemorrhagic fever

Objective: To investigate the association between IL-1 Ra variable number of tandem repeat(rs2234663), IL-6-597 GA(rs1800797), IL-6-572 GC(rs1800796) and the risk of CrimeanCongo hemorrhagic fever(CCHF) in the Turkish patients. Methods: This study included 50 patients infected with CCHF and 50 healthy controls. These variants were genotyped using polymerase chain reaction and/or restriction fragment length polymorphism method. Results: The distribution of the IL-6-572 GC genotypes and alleles varied significantly between the patients and the controls. The subjects carrying IL-6-572 GC GG genotype and G allele had increased risk of developing CCHF compared to the control group(P=0.006, P=0.014, respectively). IL-6-572 GC GC genotype was higher in the controls than the patients(P=0.006). For the triple genotype combinations, the 1/2-GC-GG genotype combination was detected more frequently in the control group than CCHF patients(P=0.016). IL-6(-572/-597) GG-GG genotype was significantly higher in the patient group(P=0.015), while the GC-GG genotype was significantly lower in the patient group(P=0.005). Additionally, the G-G haplotype was significantly higher in the patient group(P=0.042), whereas C-G was found to be significantly lower in the patients than the control group(P=0.037). Conclusions: The results of this study suggest the IL-6-572 GC variant might be genetic markers of sensitivity to CCHF in the Turkish population and may facilitate greater protection against the disease.

Establishment of a Tumor-bearing Mouse Model Stably Expressing Human Tumor Antigens Survivin and MUC1 VNTRs

The eukaryotic vectors VR1012 expressing survivin or 33 tandem repeats of human mucin 1（MUC1）（VNTRs）,namely,VR1012-S and VR1012-VNTR（VNTR=variable number of tandem repeat）,were constructed by cloning survivin and VNTR genes into VR1012,respectively.The eukaryotic vector pEGFP expressing survivin and MUC1 VNTRs fusion gene pEGFP-MS was also constructed.Mouse melanoma cell line（B16） stably expressing survivin and MUC1 VNTRs（MS ＋ B16） was established by Lipofectamine-mediated transfection of pEGFP-MS into B16 cells.EGFP expression in MS ＋ B16 cells was observed using a fluorescent microscope and survivin and MUC1 VNTRs（MS） expression was confirmed by means of Western blot analysis.A syngenic graft tumor model was generated by subcutaneous injection of MS ＋ B16 cells into C57/BL6 mice and tumor size increased rapidly with time in a cell number dependent manner.After the third immunization,mice were challenged subcutaneously with 5×l0 5 MS ＋ B16 cells.Compared with that of the negative control immunized with phosphate-buffered saline（PBS）,a significant reduction of tumor growth was observed in groups immunized with survivin plasmid DNA and MUC1 VNTRs plasmid DNA.Thus,the suppression of subcutaneous tumor was antigen-specific.This model is useful for the development of tumor vaccines targeting survivin and MUCI VNTRs.

某高级中学一起结核病聚集性疫情传播的时空序列与分子流行病学分析

目的探讨本起学校结核病聚集性疫情传播的原因、特点和规律,为持续改进学校结核病控制策略的有效实施提供科学依据。方法遵照广东省汕头市卫生健康局对本起疫情处置工作的具体要求,以指示病例为中心、以学习生活空间要素为重点、以人际交往活动要素为辅助进行密切接触者筛查,对纳入者进行结核病可疑症状检查、X线胸部检查和结核菌素(PPD)皮肤试验;对其中任何1项异常者进行IFN-γ释放试验、CT影像检查、痰涂片抗酸染色检查、分枝杆菌培养与菌种鉴定和Xpert MTB/RIF分子检测,以综合诊断结核病例;对分离培养获得的结核分枝杆菌,分析其ETRA、ETRB、ETRC、ETRE、ETRF、MIRU10、MIRU20、MIRU26、MIRU39、MIRU40、Mtub04、Mtub21、Mtub38、Oub11b、Qub26等15个基因位点的串联重复拷贝数,分析其成簇性以判断病例间的传播关系。结果(1)指示病例首次结核可疑症状出现并一直在校学习5个月后,出现双肺上叶斑片状、斑点状、条索状密度增高影和左肺上叶多发空洞(最大者约2.1 cm×1.7 cm)等病变,培养与分子检测阳性;(2)指示病例发现的3个月内,在该校相继发现其他15例学生肺结核患者,其中3例有临床症状、3例痰涂片镜检可疑阳性、9例培养阳性、7例分子检测阳性,10例与指示病例有同班上课、同舍住宿和同室晚自习等空间性密切接触,4例与指示病例有直接或间接的人际密切交往,1例则未找到明确的密切交往线索;(3)对9例患者分离培养的结核分枝杆菌进行基因分型,8例得到成功结果,15个VNTR-MIRU分型位点的拷贝数模式完全相同;(4)依据PPD皮肤试验≥5 mm判断结核感染,409例受检者中252例阳性,潜伏感染率为61.61%,潜伏感染率在指示病例班级与后续发现病例班级学生间差异有统计学意义(100%vs.56.58%,χ^(2)=30.781,P<0.001),而在男-女、学生-教职工、寄宿生-走读生、不同楼层学生、同层同侧-同层对侧学生等多组人群间则差异无统计学意义(P>0.05)。结论该起结核聚集性疫情为同一传染源传播所致,且导致了远高于一般人群的结核潜伏感染率,学校结核病控制管理缺失为其主要原因,强化学校结核病控制措施的有效执行力刻不容缓。

不同VNTR位点组合用于北京基因型结核分枝杆菌基因分型的研究

目的评价不同串联重复序列(VNTR)位点组合在北京地区北京基因型结核分枝杆菌基因分型中的应用。方法采用多位点数目可变串联重复序列分析(MLVA)技术对72株北京地区北京基因型菌株的24个VNTR位点进行分析,基因多态性分析采用BioNumerics软件。结果24个位点的多态性差异较大,位点QUB-11b(HGI 0.651)、Mtub 21(HGI0.556)和QUB-26(HGI 0.518)的多态性较高,有两个位点(MIRU 2和MIRU 24)则不具有多态性;不同VNTR位点组合(12位点,15位点,24位点)在北京基因型菌株中的分辨指数依次升高,分别为0.788,0.990,0.992,后两个组合的差异仅是由位点Mtub 29引起的。结论不同的VNTR位点在北京地区北京基因型菌株中的分辨力存在差异;推荐的基础VNTR 15位点与Mtub 29组合可作为北京地区结核分枝杆菌分子流行病学研究的一线方法。

71株浙江省结核分枝杆菌临床分离株MLVA基因分型研究

目的初步探讨浙江省结核分枝杆菌的数目可变串联重复序列(VNTR)的分型及分布特征。方法随机选取浙江省结核分枝杆菌临床分离菌株,常规培养,收集菌体,提取基因组DNA,采用聚合酶链反应(PCR)分别对VNTR位点进行检测分析,基因分型聚类分析采用BioNumerics软件。结果对71株结核分枝杆菌临床分离株的15个VNTR位点进行检测。结果显示明显的基因多态性。经基因聚类分析,可分4个基因群(Ⅰ群、Ⅱ群、Ⅲ群、Ⅳ群),58个基因型。分别为Ⅰ群占8.5%,含5个基因型,Ⅱ群占18.3%,含13个基因型,Ⅲ群占70.4%,含38个基因型,Ⅳ群占2.8%,含2个基因型。结论初步证实浙江省结核分枝杆菌VNTRs基因存在明显的多态性,至少存在4个VNTR型,主要流行型为Ⅲ型。

福建省2008-2017年人间布鲁氏菌病流行特征及分离株MLVA分型研究

目的探讨福建省人间布鲁氏菌病流行特征,并对其分离株进行分子分型,为制定预防控制策略提供依据。方法收集中国疾病预防控制信息系统中2008-2017年福建省布鲁氏菌病报告数据,进行流行特征分析;采用传统生物学鉴定和BCSP31-PCR、AMOS-PCR、MLVA-16进行布鲁氏菌分离株分子鉴定和分型,并进行聚类分析。结果福建省2008-2017年布鲁氏菌病发病呈逐年上升趋势,年均发病率0.11/10万;疫情波及福建省80%的县区,呈高度散发态势;发病高峰为4-8月;40～64岁发病数占62.7%,60～64岁组发病率最高(0.27/10万);男女比为2.50∶1,农牧民占50.7%。40株布鲁氏菌分离株分子检测结果与传统分型基本相符,为2个种(羊种和猪种)和2个生物型(羊3型和猪3型),其中羊种布鲁氏菌占绝大多数(87.5%);MLVA-16分型将其分为羊种和猪种2个种群,35株羊种菌分为28种基因型,5株猪种菌分为4种基因型,其中26种基因型为单分离株,6种基因型为共享基因型(共14株,35.0%)。同国内147株布鲁氏菌进行聚类分析显示,福建省菌株与广东和内蒙古地区存在4种共享基因型,均为羊种菌,其他部分菌株与外省菌株存在着较近的遗传距离,主要集中在panel 2B上,仅存在1～3个位点的差异。结论福建省布鲁氏菌病流行强度逐年增高,建议相关部门加强传染源的管控、对疫情高发地区的重点人群采取必要防控措施,控制其发生与流行。福建省布鲁氏菌MLVA-16分型显示高度基因多态性,提示MLVA-16可用于遗传多样性分析和分子流行病学溯源调查,可以提高布鲁氏菌病监测能力。

构巢曲霉菌基因组中的数量可变重复序列的组成和分布

利用已经公布的构巢曲霉菌基因组测序结果,对该真菌已测序基因组(30.1Mb)中的数量可变重复(VNTR)序列进行了较为系统、全面地分析。结果表明,在已经公布的基因组序列中,共有4837个以1～6个核苷酸为基序的VNTR序列(长度大于15bp,匹配值大于80%),其碱基总数占整个基因组碱基数的0.31%,平均6.2kb碱基中分布有一个大于15bp的VNTR。其中数量最多的五碱基VNTR,数量达到1386个,其次为六碱基VNTR(1228个),三碱基VNTR(1199个),这3种VNTR总数达3813个,占VNTR总数的78.8%。数量最少的是二碱基VNTR,只有144个。在9541个开放阅读框架(ORF)中的VNTR总数为1683个,共分布于1356个ORF中。其中只有1个VNTR的ORF为1117个。与其他生物内VNTR的分布类似,在基因编码区中,以三碱基VNTR和六碱基VNTR占绝对优势,在阅读框架中的VNTR中分别占到58.0%和52.9%。编码区的三碱基、六碱基VNTR分别为该菌基因组中相应VNTR总数的约44.4%和38.6%。由于编码区的碱基数占基因组碱基数的59.3%,所以这两种长度的VNTR在编码区中的密度略低于基因组中的平均密度。在编码区上下游300bp调控区,除在编码区数量较多的三碱基VNTR和六碱基VNTR外,其他各种长度的VNTR的比例都超过了10%。可见300bp的上下游调控区域是单碱基、二碱基、四碱基。

220株结核分枝杆菌北京临床分离株的基因分型研究

目的了解结核分枝杆菌北京临床分离株的基因分型种类和特征,为结核病防治研究提供基础科学依据。评价两种分型方法在北京地区结核病分子流行病学中的应用。方法收集北京市结核分枝杆菌临床分离株,应用间隔区寡核苷酸分型(Spoligotyping)及多位点可变数目串联重复序列分析(MLVA)二种分型方法进行基因分型,结果聚类分析采用BioNu-merics(Version5.0)软件。结果共在北京地区收集到220株结核分枝杆菌临床分离株。采用Spoligotyping分型方法,220株菌可分为2个基因群,即北京家族(Beijing family)和非北京家族(non-Beijing family),20种基因型。其中北京家族含有202株菌,占91.82%。北京家族菌株中,典型北京家族菌株占95.05%(192/202),非典型北京家族占4.95%(10/202)。北京家族菌株的耐药率为22.55%(22/98),非北京家族菌株的耐药率为16.67%(1/6),两者间的差异无统计学意义(fisher analysis,P=0.5835>0.05)。采用MLVA分型方法,202株菌可分成5个基因群59种基因型,主要为Beijing family、China2和China3,分别占91.37%、2.73%和4.55%。结论北京地区结核分枝杆菌具有明显的基因多态性,其主要流行型为北京家族。北京家族菌株与耐药性无明显相关性。

数目可变串联重复序列用于江苏省168株结核分枝杆菌菌株的基因分型研究

目的:初步了解江苏省结核分枝杆菌临床分离菌株的数目可变串联重复序列(Variable number of tandem repeats,VNTR)的基因型及VNTR基因分布特征。方法:在苏南、苏中、苏北三个地区13个结核病定点诊疗单位收集临床分离菌株。选取结核分枝杆菌基因组中11个具有明显多态性特征的VNTR位点,通过PCR扩增,2%琼脂糖凝胶电泳和Bio Numerics(Version 3.0)软件进行结核菌株VNTR的多态性和聚类分析。结果:共收集到168株结核分枝杆菌。不同的菌株在同一位点上具有多态性。共分为10个基因型(Ⅰ,Ⅱ～Ⅹ型),其中以Ⅷ型为主要基因型,占75.0%、其次为Ⅱ型5.4%、Ⅳ型4.2%、Ⅶ型9.5%。不同地区之间,苏南、苏中、苏北三个地区也都以Ⅷ型为主要基因型,分别占各地区的83.5%,57.1%,76.6%;但是苏南、苏中、苏北三地的Ⅱ、Ⅲ、Ⅳ、Ⅶ型菌株分布在各地菌株中的比例有差异显著性((2=54.710,P<0.0001),Ⅱ型以苏中为主(11.9%)、Ⅲ型以苏南为主(3.8%)、Ⅳ型以苏南、苏北为主(5.1%与6.4%)、Ⅶ型以苏中为主(31.0%)。结论:江苏省的结核分枝杆菌具有明显的多态性,以Ⅷ型为主要流行型,不同地区间同样以Ⅷ型为主要流行型,但菌型分布存在一定的差异。

云南省剑川县鼠疫疫源地分离菌株基因组多态性

目的阐明云南剑川野鼠疫源地鼠疫菌种群的演化及其与云南省其他疫源地菌株之间的遗传进化关系。方法按不同时间、不同疫点、不同分离源,随机选取24株剑川分离鼠疫菌进行研究;采用差异区段(DFR)、规律成簇的间隔短回文重复序列(CRISPRs)及多位点可变数目串联重复序列分析(MLVA)进行基因组多态性分析;以DFR+CRISPRs聚类分析划分簇,再以MLVA26对簇进行聚类分析。结果24株剑川菌株中,20株菌聚为一个簇(剑川野鼠鼠疫簇);2017年疫情分离3株菌位于丽江野鼠鼠疫簇,与丽江菌株(2017LZ)存在2个位点的差异(N2577,M23);剩余1株为一个独立株;50年代菌株与其邻近的80年代菌株间位点差异未超过3。结论剑川原野鼠疫源地的发现时间可往前推至上世纪50年代,同时疫情还波及过家鼠和人群;剑川县境内存在2种类型的野鼠疫源地,其鼠疫防控具有复杂性,应继续加强鼠间鼠疫的动态监测。

老年住院患者肺炎克雷伯菌临床分离株多位点可变数目串联重复序列基因分型特征分析

目的探讨老年住院患者肺炎克雷伯菌分离株多位点可变数目串联重复序列分析的基因分型特点。方法收集解放军总医院南楼临床部老年住院患者肺炎克雷伯菌临床分离菌株184株,提取基因组DNA,采用多重PCR和毛细管电泳对7个位点进行分析,利用Bio Numerics 5.1软件进行聚类分析。结果 184株肺炎克雷伯菌临床分离株共产生139种基因型,呈现明显的基因多态性;聚类分析结果显示,全部菌株可分为3个基因群,其中Ⅰ群包含93.06%的菌株。结论本医疗中心老年住院患者的肺炎克雷伯菌临床分离株具有明显的优势菌群,其感染来源仍以院内感染为主。