霍乱弧菌16SrRNA基因分型研究

利用１６ＳｒＲＮＡ基因探针分析了某地不同时期分离的８８株霍乱弧菌经ＢｇｌⅠ消化的１６ＳｒＲＮＡ基因限制性酶谱,结果发现各菌株的杂交片断范围为２－１２ｋｂ,每个菌株有６－９条杂交带不等。８８株霍乱菌株可分为９个１６ＳｒＮＲＡ基因型（ＲＴ）,其中ＥＶＣ可分为６个ＲＴ,ＶＣＯ１３９分为３个ＲＴ,认为霍乱流行菌株基因型的变迁可能是引起新一次流行的原因。结合ｃｔｘＡＢ基因检测结果,我们认为ＶＣＯ１３９与ＥＶ     

霍乱弧菌O1血清群产毒株重复基因分析

目的对霍乱弧菌O1血清群产毒株基因组中重复基因数量分布、多样性及两种生物型中重复基因的差异进行分析。方法选择300株引起第6次和第7次大流行的O1群古典型和埃尔托型生物型菌株,使用BLAST和MCL等软件分析重复基因数量分布及两种生物型差异的重复基因;使用序列两两平均距离作为多样性的度量方法分析重复基因的遗传多样性以及在两种生物型之间存在遗传多样性差异的重复基因。结果每株菌平均包含242.63个同源基因簇,其中每株埃尔托型和古典型菌株分别平均包含244.85和209.74个。 筛选到13个在埃尔托生物型与古典生物型之间拷贝数存在显著差异同源基因簇,功能主要包括阴离子、氨基酸等物质的转运、定位、跨膜转运等。 对存在重复基因的同源基因簇的遗传多样性分析,发现在两种生物型之间多样性存在差异的同源基因簇50个,这些基因簇的功能主要包括阴离子、氨基酸、有机底物等转运、定植、底物特异的跨膜转运活性等。结论霍乱弧菌重复基因的进化有一定的功能倾向性,其特征解析对于理解其基因组适应性进化、菌株多样性及两种生物型基因组差异进化提供了新依据。     

乳胶凝集法快速检测血清肌红蛋白

乳胶凝集法快速检测血清肌红蛋白卫生部北京医院检验科（１００７３０）艾桂平，杨振华卫生部临床检验中心徐建平肌红蛋白（Ｍｂ）是存在于人心肌、骨骼肌中的呼吸蛋白，在发生心肌梗塞或肌肉损伤时，血液中大量出现。特别是在急性心肌梗塞（ＡＭＩ）时，Ｍｂ的出现早于各...     

2株与霍乱弧菌O139诊断血清发生凝集的麦氏弧菌鉴定

目的对2株疑似O139群霍乱弧菌进一步鉴定,以排除或确诊霍乱。方法以试管凝集和交叉吸收实验复核玻片凝集结果,用生化实验和BIOFOSUN 微生物鉴定系统进行生化鉴定,PCR技术检测O139霍乱弧菌特异性脂多糖基因（LPS）。结果玻片凝集阳性,试管凝集1株菌可达原诊断血清效价的1/4,1株菌达1/2,交叉吸收后均证实为非特异性凝集;未检出LPS基因;系统生化鉴定判定为麦氏弧菌。结论2株菌不是O139霍乱弧菌,而是麦氏弧菌。     

致血流感染非O1群非O139群霍乱弧菌的鉴定及毒力基因检测

目的对襄阳市中心医院分离1株疑似霍乱弧菌进行鉴定及药物敏感性试验,并检测其主要毒力基因。方法利用MicroScan WalkAway 40鉴定仪进行生化鉴定及药物敏感性试验,玻片凝集法确定其血清型别,应用PCR及测序技术分析其16SrRNA基因;PCR检测其6个毒力基因。结果经鉴定,该株疑似霍乱菌株为非O1群非O139群霍乱弧菌,经16SrRNA分析与美国国家生物技术信息中心数据库中霍乱弧菌相似性达100%。药敏试验结果显示该菌对氨苄西林、氯霉素、甲氧苄啶-磺胺甲（口恶）唑、四环素均敏感,毒力基因检测rtxC和toxR阳性,tcpAET、ctxA、hlyA、tcpACL阴性。结论该株疑似霍乱弧菌为非O1群非O139群霍乱弧菌,其致病与rtxC、toxR毒力基因有关。     

霍乱弧菌01群和0139群快速检测的研究进展

本文综述了霍乱弧菌01群和0139群快速检测的研究进展，主要包括免疫学检测，基因探针检测，以及PCR检测。     

应用基因测序方法检测哮喘儿童的PTEN基因突变

目的分析10号染色体上缺失的磷酸酶与张力蛋白同源基因（phophatase and tensin homolog deleted onchromosone 10,PTEN）突变与儿童哮喘的相关性,及其与外周血嗜酸性粒细胞（eosinophils,EOS）的关系,进一步探讨儿童哮喘的发病机制,并为其新治疗方法的研发提供一定理论依据。方法设定30例哮喘患儿（临床缓解期）为哮喘组,30例健康儿童为对照组。均于清晨空腹抽取外周静脉血,制备血涂片后提取全基因组DNA。对其PTEN基因第5、第8外显子（exon 5,exon 8）进行基因测序分析,同时进行外周血EOS计数。计量资料采用χ2 test,计数资料采用t检验进行统计分析。结果 83.3%（25/30）哮喘组儿童有PTEN基因第8内含子（intron 8）突变,对照组无基因突变。外周血EOS计数在哮喘组明显高于对照组,差异显著（P〈0.05）;发生基因突变的哮喘患儿外周血EOS计数高于无突变的哮喘患儿,差异显著（P〈0.05）。结论哮喘儿童绝大部分存在PTEN基因第8内含子突变,其与EOS的数量相关,PTEN基因可能通过抑制EOS的活化和趋化在哮喘发病中发挥作用。     

协同凝集试验检测O1群霍乱弧菌

目的建立一种快速简便的O1群霍乱弧菌检测方法。方法将抗O1群霍乱弧菌多价单克隆抗体包被到金黄色葡萄球菌CowanI株上制成SPA菌体试剂,建立起协同凝集试验(COA),检测粪便标本6h培养物中的霍乱弧菌。结果COA检测的敏感性为4×106CFU/ml,检测过程为5min。92份临床标本的测定结果与培养法相比较,COA的特异性、敏感性、诊断符合率分别是100%、95.5%、97.8%。结论COA简便、快速,可作为一种新的霍乱弧菌快速诊断方法。     

超声造影联合血清学在卵巢癌早期筛查中的应用研究

目的 探讨超声造影指标联合血清学糖类抗原125 (CA125)、癌胚抗原(CEA)、人附睾分泌蛋白4(HE4)在卵巢癌早期筛查中的应用价值.方法 选取卵巢占位性病变患者105例,均行超声造影检查和血清CA125、CEA、HE4检查,分析超声造影联合血清CA125、CEA、HE4对卵巢癌的诊断效能.结果 相比良性组,卵巢...     

全身骨显像与PSA 联合检测在前列腺癌中的临床应用

目的 为探讨放射性核素骨显像与血清前列腺特异抗原（ＰＳＡ）深度联合检测在前列腺癌（ＰＣａ）中的临床意义。方法 对６３例ＰＣａ、２４例骨显像分级２级以上患者治疗后行骨显像及血清ＰＳＡ含量测定结果进行了回顾性分析。结果 ＰＣａ患者血清ＰＳＡ测定阳性率高,对临床诊断具有参考价值;而骨显像对于临床分期具有重要指导作用;２４例显像分级２级以上的患者骨转移程度与血清ＰＳＡ含量间有显著相关性。结论 骨显像与血清     

彩超结合血浆CA125在早期子宫内膜癌诊断中的应用

目的评价子宫内膜癌血流特点及其与血浆CA125值相互关系;利用血流特点结合血浆CA125值诊断早期子宫内膜癌。方法检测25例子宫内膜癌（其中早期11例）、35例内膜息肉、11例黏膜下肌瘤患者治疗前病灶血流特点及血流阻力指数（RI）;免疫放射定量测定患者治疗前后血浆CA125值。结果子宫内膜癌患者病灶内见较丰富彩色血流、分布极紊乱、RI为中低阻,且血浆CA125值含量较高,二者间存在明显负相关性。内膜息肉患者可显示息肉根部增粗的血管、RI较高,且血浆CA125值含量较低。黏膜下肌瘤患者可见环状彩色血流信号、RI呈中高阻,且血浆CA125值含量较低。结论彩超结合血浆CA125值为早期子宫内膜癌的诊断提供有价值信息。     

柱凝集抗球蛋白试验在疾病诊断中的应用

目的 探讨柱凝集抗球蛋白试验在诊断自身免疫性溶血性贫血（AIHA）等疾病和交叉配血中的应用。方法 采用传统试管法（CTT）和柱凝集法（CAT）对547例样本同时进行抗球蛋白试验。交叉配血试验前柱凝集法直接抗球蛋白试验（CAT-DAT）初筛阳性85例,同时做手工聚凝胺法（MPT）和CAT法交叉配血试验。结果 CTT、CAT两种方法的直接抗球蛋白试验（DAT）阳性率分别为6．8％和25．2％。CTT阳性结果较为集中,主要见于新生儿溶血病、AIHA;而CAT阳性结果较为分散,除常见的新生儿溶血病、AIHA外,尚有再生障碍性贫血、Evan’s综合征、败血症、系统性红斑狼疮、腺病毒感染、肺炎、先天性心脏病等。51例经临床确诊的AIHA患者,测定CTT-DAT阳性24例,而CAT-DAT均为阳性,两种方法测定AIHA患者DAT的差异有显著性。CAT-DAT阳性样本用CAT法交叉配血次侧均为阳性,而MPT法只有20％为阳性;MPT法次侧阳性用CAT法检测相应结果均为强阳性,这两种交叉配血方法的差异有显著性。结论 与传统试管法相比,柱凝集抗球蛋白试验在诊断AIHA时有更高的敏感性;与手工聚凝胺法相比,柱凝集抗球蛋白试验交叉配血亦有更高的敏感性。     

OPRM1基因PCR产物和测序产物三种纯化方法的比较

目的 探讨一种快速、简便、效果好的OPRM1基因PCR产物和测序产物纯化方法,建立稳定的DNA测序技术平台.方法 采用乙醇/醋酸钠、过柱和SAP-Exonuclease Ⅰ结合BigDye Xterminator Purification Kit 3种纯化方法,对50份OPRM1基因PCR产物和测序产物进行纯化效果的比较.结果 SAP-Exonuclease Ⅰ法对PCR反应产物进行纯化的回收率显著高于乙醇/醋酸钠和过柱法,分别为0.86±0.01,0.48±0.08,0.65±0.01(t=32.5,P＜0.001;t=86.1,P＜0.001);BigDye Xterminator Purification Kit法对测序产物进行纯化成功率显著高于乙醇/醋酸钠和过柱法,分别为100％,62％和84％(x2=23.46,P＜0.001;x2 =8.70,P=0.003).结论 SAP-Exonuclease Ⅰ结合BigDye Xterminator Purification Kit是一种快速、简便、效果好的测序纯化方法.     

Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement–negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel–based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.The detection of recurrent chromosomal rearrangements by cytogenetics was one of the earliest clinical molecular oncology assays and continues to play a major role in cancer diagnosis and prognosis.^1,2 Although translocations in the clinical laboratory are generally detected by cytogenetics, fluorescence in situ hybridization (FISH), or RT-PCR, studies have demonstrated that they may also be detected by next-generation sequencing (NGS) of DNA or RNA.^3–5 DNA-level translocations can be detected in particular areas of interest by first performing hybrid capture enrichment to target one or both partner genes in a translocation, followed by NGS.^4,6 NGS-based translocation detection has several advantages over conventional clinical laboratory methods, such as the ability to precisely define the breakpoint region, detect cryptic rearrangements and unknown partner genes, and run in parallel with gene mutation detection.Chromosomal rearrangements are detected in the clinical laboratory by routine cytogenetics, FISH, or RT-PCR; however, these methods have limitations. Cytogenetic studies, including chromosome analysis and metaphase FISH, require actively dividing cells, which can be especially difficult to obtain from solid tumors. In addition, chromosome analysis is of limited resolution, particularly in oncology specimens, and is therefore insensitive to cryptic and complex rearrangements.^5,7,8 Some rearrangements can be assayed via RNA-based RT-PCR methods, but this approach is less useful for translocations with a large number of partner genes or those with potentially diverse breakpoints.^9,10 FISH is among the most commonly used laboratory methods for the detection of chromosomal rearrangements and offers high sensitivity and the ability to test routine interphase, formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, FISH relies on highly trained individuals to score rearrangements by fluorescent microcopy and is an inherently low-resolution method that may be confounded by complex, multiway rearrangements and may require numerous probes to fully elucidate translocation partners for promiscuous genes, such as KMT2A.^5,10 Finally, FISH results are generally difficult to validate by orthogonal methods, outside less sensitive cytogenetic assays.Two of the most commonly tested translocations in the clinical laboratory are for rearrangements of the anaplastic lymphoma kinase gene, ALK, in non–small cell lung cancer and of the mixed-lineage leukemia gene, KMT2A (formerly known as MLL), in acute leukemia. The EML4-ALK fusion results from an inversion event on chromosome 2p that generally causes an in-frame fusion of EML4 exons 1 to 13 to ALK exons 20 to 29, producing an aberrant fusion gene with constitutive kinase activity, sensitive to crizotinib.^11–14 The occurrence of ALK fusions and other common lung cancer gene mutations in KRAS and EGFR are generally considered to be mutually exclusive, arguing that these tumors represent a distinct subset of lung cancers.¹⁵ Although not pharmacologically targetable, KMT2A rearrangements are of diagnostic and prognostic significance in acute leukemias, including both acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).^16,17 KMT2A rearrangements can be readily detected by FISH using break-apart probes; however, elucidation of the translocation partner gene may be difficult because >100 have been identified.^10,18NGS has had a tremendous effect on cancer discovery and is now becoming routine in the clinical molecular oncology laboratory.^3,19–21 NGS allows for the cost-effective, simultaneous evaluation of numerous sequence variants as part of focused clinical oncology panels or whole exomes. We and other groups have previously found that a range of DNA variants, including translocations, insertions or deletions, and copy number variants, can be detected from targeted NGS data and that it is possible to identify DNA-level breakpoints with single-nucleotide precision.^4,22,23 However, to be useful in the clinical setting, a thorough evaluation of the sensitivity and specificity of structural variation (SV) detection by NGS compared with standard methods is required. Given that numerous potential translocations can be evaluated by NGS simultaneously as part of a larger NGS cancer panel, for little to no additional cost, such methods could provide a significant savings for laboratories that perform multiple single-gene tests and multiple FISH assays on oncology specimens.We present a comprehensive evaluation of targeted translocation detection by NGS in the clinical laboratory by comparing four publicly available translocation detection tools (including the laboratory derived ClusterFAST) on targeted NGS data from 13 cases with ALK or KMT2A rearrangements (six lung carcinomas and one anaplastic large cell carcinoma with ALK rearrangements; six leukemias with KMT2A rearrangements) and 77 cancers negative for ALK and KMT2A rearrangements by FISH. We found that translocations can be reliably detected at the DNA level by targeted NGS panels and that such methods offer sensitivity and specificity similar to that of routine FISH with the advantage of single-nucleotide breakpoint resolution. Further, we examine approaches to designing capture probes for targeted NGS evaluation, evaluate the minimal coverage levels necessary to detect translocations, and explore methods to reduce false-positive translocation reports.     

血清NGAL与肾功其它指标联合检测在原发性高血压肾病早期诊断中的应用

目的 检测血清中性粒细胞明胶酶相关脂质运载蛋白(NGAL)浓度,探讨其与胱抑素 C(Cys-C)、视黄醇结合蛋白(RBP)、尿素(BUN)和肌酐(Cr)联合测定在原发性高血压早期肾脏损害中的应用价值。方法 选择2014年8月～2016年3月于陕西省人民医院就诊并经确诊的128例原发性高血压患者,将其分为单纯原发性高血压组、原发性高血压并发肾病组和原发性高血压并发肾衰组,同时选择100例健康体检者作为对照组。采用日立7170A全自动生化分析仪对各组血清NGAL与Cys-C,RBP,BUN和Cr水平进行检测,其结果进行统计学分析。结果 各组血清NGAL,Cys-C,RBP,BUN和Cr的检测结果中,单纯原发性高血压组与正常对照组之间差异均无统计学意义(P均>0.05); 原发性高血压并发肾病组与正常对照组、单纯原发性高血压组比较差异均有统计学意义(P均<0.05); 原发性高血压并发肾病组和原发性高血压并发肾衰组血清NGAL与正常对照组、单纯原发性高血压组差异均有统计学意义(P均<0.01); 而原发性高血压并发肾病组NGAL与Cys-C之间差异无统计学意义(P>0.05)。各组NGAL,Cys-C,RBP,BUN和Cr检测异常率方面,原发性高血压并发肾病组均显著高于正常对照组和单纯原发性高血压组,差异有统计学意义(P均<0.01),但BUN,Cr远低于原发性高血压并发肾病组NGAL的异常率(P<0.01)。而原发性高血压并发肾衰组的NGAL,Cys-C,RBP,BUN和Cr异常率略有差异,但差异均无统计学意义(P>0.05)。结论血清NGAL的检测在原发性高血压肾病早期诊断中具有重要的意义,如果联合肾功其它指标的检测可为临床诊断和评估原发性高血压早期肾脏损害以及损害的程度提供重要依据。