PCR和毛细管电泳技术检测沙门氏菌等5种病原菌的方法研究

目的采用多重PCR技术检测结合毛细管电泳成像技术建立快速检测沙门氏菌、志贺氏菌、副溶血性弧菌金黄色葡萄球菌和大肠杆菌O157的方法。方法根据沙门氏菌hilA基因、志贺氏菌ipaH基因、副溶血性弧菌TDH基因、金黄色葡萄球菌的pSa-442 Sau3AI fragment基因及大肠杆菌O157的rfbE基因设计异性特PCR引物,被检样品经4 h振荡培养后金属浴裂解制备DNA模板,使用全自动毛细管电泳核酸检测系统分析PCR扩增产物。结果在580 bp、423bp、257 bp、245 bp和194 bp处分别出现预期的特异性DNA条带,且无非特异扩增条带出现。敏感性试验显示在模拟标本中的检测灵敏度,沙门氏菌为101-2cfu/ml、志贺氏菌为101cfu/ml、副溶血性弧菌为102cfu/ml、金黄色葡萄球菌为102cfu/ml、大肠杆菌O157为101cfu/ml。结论该方法操作方便、特异性和灵敏度高、分辨率高,可同时完成5种病原菌的检测,在公共卫生突发事件中具有实用价值。     

应用多重PCR技术鉴定我国6个旋毛虫地理株的研究

目的 对我国猪源旋毛虫地理株进行鉴定和分类。 方法 根据旋毛虫rDNA扩展片段V区（expansion segment V region,ESV）和内转录间隔区（internal transcribed spacers,ITS）ITS1和ITS2基因序列合成5对特异性引物,以旋毛虫（Trichinella spiralis,T1）、乡土旋毛虫（T. nativa,T2）、布氏旋毛虫（T. britovi,T3）、伪旋毛虫（T. pseudospiralis,T4）及纳氏旋毛虫（T. nelsoni,T7）的国际参考株作为对照,应用多重PCR对我国6个猪源旋毛虫地理株（河南、云南、哈尔滨、黑龙江同江、湖北及天津）进行鉴定,并观察影响多重PCR扩增的因素。 结果 我国6个猪源旋毛虫地理株多重PCR扩增结果显示,均具1条与T1相同的条带（173 bp）。应用多重PCR对单条旋毛虫幼虫、不同保存条件的旋毛虫幼虫及含幼虫的新鲜小鼠肌肉提取液进行扩增,均得到173 bp的特异性条带。 结论 经多重PCR鉴定我国6个猪源旋毛虫地理株均为T1。     

莫西沙星耐药的艰难梭菌多重聚合酶链反应检测和基因分型

目的：建立一种多重 PCR 方法用于莫西沙星耐药的艰难梭菌鉴定和初步基因分型。方法根据艰难梭菌 slpA 可变区间核苷酸序列的差异设计5种 slpA 基因型（gr、hr、fr、gc08和078）的特异性 PCR 引物,同时加入检测艰难梭菌管家基因磷酸甘油醛异构酶基因 tpi 的种特异性引物,构建多重PCR 方法;利用9种肠道常见的正常或致病菌验证多重 PCR 方法的特异性,利用46株分属于11个slpA 基因型的艰难梭菌参考菌株来验证方法的检测和分型能力;利用建立的多重 PCR 方法检测39株莫西沙星耐药的临床菌株,以 slpA 测序分型法为参照方法,评估该方法的临床实用性。结果多重PCR 检测9种肠道常见的正常或致病菌 tpi 和5种 slpA 基因型均为阴性;46株艰难梭菌参考菌株 tpi均为阳性,36株分属于5种靶 slpA 基因型（gr、hr、fr、gc08和078）的菌株被正确分型,10株分属于其他6种基因型的参考菌株均无法分型。39株莫西沙星耐药的艰难梭菌临床菌株 tpi 均为阳性,32株检出具体基因型,其中22株为 slpA 基因型 gc08,6株为 hr,2株为 fr,2株为078,与 slpA 测序分型结果一致;7株多重 PCR 无法分型,slpA 测序分型结果显示其基因型均不包含在多重 PCR 分型范围内。结论成功建立一种简单、快捷、临床实验室适用的艰难梭菌检测,且能够分辨出5种 slpA 基因型的多重PCR 方法;莫西沙星耐药的艰难梭菌主要为 slpA 基因型 gc08。     

Novel characterization of a breakpoint in F8: an individualized approach to gene analysis when PCR and MLPA results contradict

Haemophilia A is an X‐linked bleeding disorder caused by heterogeneous mutations in the F8 gene. Two inversion hotspots in intron 22 and intron 1, as well as point mutations, small insertions and deletions in the F8 gene account for causal mutations leading to severe haemophilia A. Rarely, novel molecular mechanisms lead to a haemophilia A phenotype which cannot be completely characterized by routine molecular diagnostic methods. Here, we characterized the molecular abnormality in a boy with a severe haemophilia A phenotype. On investigation by PCR and DNA sequencing, exon 18 of F8 repeatedly failed to amplify. However, analysis by multiplex ligation‐dependent probe amplification demonstrated the presence of exon 18 sequence, suggesting a more complex rearrangement than a single exon deletion. The analysis of exon 18 and its flanking regions by inverse PCR revealed a complex mutation comprising insertions of extragenic sequences from Xq28 along with a partial duplication of exon 18. Based on the successful analysis and characterization of the familial breakpoint, we developed a PCR‐based diagnostic approach to detect this defect in family members in whom no diagnostic test could be offered until this time.     

A sensitive and a rapid multiplex polymerase chain reaction for the identification of Candida species in concentrated oral rinse specimens in patients with diabetes

Objectives: Oral candidiasis is being frequently recognized in patients with diabetes, and is associated with multiple pathogens including Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis. The aim of this study was to evaluate a usefulness of a Multiplex Polymerase Chain Reaction as a rapid diagnostic tool for identification of four oral Candida pathogens in patients with diabetes.

Materials and methods: A multiplex PCR was optimized to identify four Candida species in concentrated oral rinse samples. Common reverse primer, ITS4 and four species-specific forward primers targeting ITS1 and ITS2 regions of yeast genome were used. Species-specific single amplicon were detected by agarose gel electrophoresis. Performance efficacy of multiplex PCR was compared with phenotypic identification.

Results: Out of 100 oral rinse samples, 72 were culture positive and of these 43 were at risk of oral Candida infection (>600cfu/ml). Multiple Candida species including C. albicans, C. parapsilosis and C. tropicalis were identified in 22 samples which had risk of oral Candida infection. In total, 85 patients were positive for Candida by multiplex PCR and of them 49 had multiple Candida species. All 43 colonized specimens were also positive by multiplex PCR. C. albicans was the most predominant organism (75/85) followed by C. parapsilosis (47/85), C. tropicalis (17/85) and C. glabrata (6/85). In specimens with multiple species, the two most common organisms were C. albicans and C. parapsilosis. Multiplex PCR yielded a sensitivity of 10 Candida cells/ml of oral rinse sample.

Conclusions: Multiplex PCR is found to be rapid, sensitive and specific than phenotypic identification methods in discriminating multiple Candida species in oral rinse specimens.     

Analysis of isodicentric Ph chromosomes in chronic myeloid leukemia blast crisis北大核心CSCD

Objective To explore the genetic and clinical characteristics of isodicentric Ph chromosomes [idic(Ph)] in lymphoid blast crisis of chronic myeloid leukemia (CML-BLC). Methods Bone marrow aspirates of 2 patients withCML-BLC were analyzed by R banding after 24 hours of culturing. Genomic copy number variations (CNV) wereanalyzed by single nucleotide polymorphism array (SNP array) in case 1. The results were confirmed withfluorescence in situ hybridization (FISH). Variations of acute lymphoblastic leukemia-related genes includingCDKN2A/AB and PAX5 were detected by multiplex ligation-dependent probe amplication (MLPA). Results Deletionsand duplications on derivative chromosome 9 detected by FISH were confirmed by SNP array analysis. Thedistances between the BCR/ ABL fusion signals on the idic(Ph) chromosomes in the two patients have differedgreatly. The idic(Ph) in the second patient was supposed to be formed by two Ph chromosomes joined at their qterminals, where as the idic(Ph) in the first patient have been shown to be fused at the satellite regions oftheir p arms. Conclusion The idic(Ph) chromosomes presented in CML-BLC may predict resistance to Imatinib andresponse to Dasatinib.     

Analysis of the cause of pregnancy failure with combined MLPA assay for subtelomeric regions and ultrasonography北大核心CSCD

Objective To explore the value of multiplex ligation-dependent probe amplification (MLPA) for thedetection of chromosome abnormalities in miscarriage tissues, and to correlate the result with ultrasoundfindings. Methods A total of 421 cases of spontaneous abortions and fetal deaths were detected with the MLPAmethod. Results Among the 421 samples, 232 (55. 11%) had an abnormal MLPA result. For the 286 cases derivedfrom < 13 weeks pregnancy, 206 (72.03%) were abnormal. For the 49 cases from 14 ~ 19 weeks pregnancy, 14(28. 57%) were abnormal. For the 86 cases derived after 20 weeks pregnancy, 12 (13. 95%) were abnormal. Amongthe 117 cases with abnormal ultrasound findings, 33 cases (28. 21%) had an abnormal MLPA result, 28 out of the33 cases were numerical chromosome abnormality, 4 cases were chromosome microdeletion and/or micro duplication, 1 case had both numerical abnormality and microduplication. For those with abnormal ultrasound findings for the neck region, fetal edematous syndrome, multiple malformations and digestive system, the detection rates forMLPA were 71. 4% , 58. 8% , 37. 8% , and 9.1% , respectively. For those with abnormal finding of cardiacsystem, nervous system, face, skeletal system and urinary system, none was found with positive results of MLPA. Conclusion Numerical chromosomal abnormalities account for the majority of cases with spontaneous abortion.With the increase of gestational age, the occurrence of chromosomal abnormalities gradually declines. Combinedultrasound and MLPA assay can improve the detection rate and accuracy for chromosomal abormalities.     

耐甲氧西林金黄色葡萄球菌SCCmec基因分型及耐药谱分析

目的 研究耐甲氧西林金黄色葡萄球菌(MRSA) SCCmec基因分型情况,并对其耐药谱进行分析.方法 收集从临床标本分离出的MRSA 91株,应用多重PCR法对MRSA进行SCCmec基因分型,采用MicroScanWalk-Away-40全自动微生物鉴定药敏分析仪进行细菌的鉴定及药敏试验,部分药敏试验采用K-B法.结果 91株MRSA中SCCmecⅡ型72株,占79.1％,SCCmecⅢ型16株,占17.6％,未分型3株,占3.3％,未见SCCmec Ⅰ型及SCCmecⅣ型;ICU中MRSA所占比例最多,为SCCmecⅡ型51.4％、SCCmecⅢ型37.5％,2种SCCmec基因型的MRSA对万古霉素、磺胺甲噁唑/甲氧苄啶、利奈唑胺、喹奴普汀/达福普汀的耐药率为0,对氯霉素的耐药率分别为11.1％和12.5％,对利福平的耐药率分别为8.3％和37.5％,其余均呈高水平耐药,2种SCCmec基因型的MRSA均表现为多药耐药.结论 临床分离的MRSA以SCCmecⅡ型为主,且对抗菌药物呈多药耐药.     

Multiplex ARMS PCR to Detect 8 Common Mutations of ATP7B Gene in Patients With Wilson Disease

Background

Wilson disease is a rare disorder of copper metabolism due to mutation in ATP7B gene. Proper counseling of patients with Wilson disease, and their families necessitates finding mutation in ATP7B gene. Finding mutations in ATP7B gene with 21 exons, and more than 500 mutations is expensive and time-consuming.

Objectives

The aim of this study was to provide a simple multiplex amplification refractory mutation system PCR (M-ARMS-PCR) for screening eight common mutations in ATP7B gene.

Patients and Methods

Two sets of ARMS mutant and normal specific primer pairs were designed for genotyping of p.R778L, p.R969Q, p.H1069Q, and p.3400delC mutations as Set 1 and p.W779G, c.3061-1G > A, p.I1102T, and p.N1270S mutations as Set 2. The Multiplex ARMS assay was then subsequently tested in 65 patients with Wilson disease with known and unknown ATP7B mutations.

Results

Using these two sets, we identified H1069Q mutation in four patients, c.2335T > G mutation in three, c.3061-1G > A splice site mutation in five, c.3305T > C mutation in one, and c.3809A > G mutation in two patients.

Conclusions

The Multiplex ARMS assay used in this study can be an efficient, reliable, and cost effective method as a primary screen for patients with Wilson disease.