桂西地区幽门螺杆菌对克拉霉素的耐药性分析

目的 分析桂西地区幽门螺杆菌(Helicobacter pylori,HP)菌株对克拉霉素的耐药情况,并探讨Hp对克拉霉素耐药与23S rRNA基因点突变的关系.方法 分离培养Hp,纸片扩散法进行药敏实验,PCR方法扩增23S rRNA基因,用聚合酶链反应.限制性片段长度多态性(PCR-RFLP)检测克拉霉素耐药菌株的点突变,同时进行基因测序确定突变位点.结果 桂西地区Hp菌株对克拉霉素耐药率为22.2%(28/126);PCR-RFLP检测10株对克拉霉素耐药的Hp,均存在23S rRNA基因的A2143G、A2144G点突变,10株敏感菌株均无23S rRNA的点突变,基因测序显示耐药菌株有A2143G、A2144G突变,其他位置未发现突变.结论 桂西地区Hp菌株对克拉霉素耐药率(22.2%)略高于北京、上海等地区;Hp的23S rRNA基因A2143G、A2144G点突变与克拉霉素的耐药相关.     

两个非综合征型耳聋家系中线粒体DNA 12S rRNA基因A827G突变

目的探讨线粒体DNA（mitochondrial DNA，mtDNA）12S rRNA基因与中国人非综合征型遗传性耳聋的关系。方法对两个母系遗传性的非综合征型耳聋家系中20名成员及32例散发耳聋患者外周血DNA进行12S rRNA、tRNA^ser（UCN）以及GJB2基因PCR扩增，产物通过限制性片段多态性分析及基因测序，进行突变检测和分析。结果所有研究对象的基因区域均扩增成功。12S rRNA全序列测定发现两家系中所有受检的母系成员（包括12例耳聋患者）均存在nt827A→G转换，并表现为同质性突变。而非母系成员该位点序列正常。32例散发耳聋中有1例A827G突变阳性。未检测到GJB2基因、tRN^ser（UCN） A7445G及12S rRNA A1555G突变。结论再次验证了mtDNA 12S rRNA基因突变在母系遗传性非综合征型耳聋发病中的重要性。首次发现mt DNA 12S rRNA nt827A→G转换是导致两个中国家系耳聋遗传易感性的分子基础。     

线粒体12S rRNA基因突变与2型糖尿病

目的 观察线粒体12S rRNA 1310、1438及1442位点在中国汉族2型糖尿病患者群体中的突变情况,同时筛查该区域与2型糖尿病发生有关的突变。方法 采用PDR－SSCP及PCR产物直接测序等技术对86例2型糖尿病患者及70名正常对照个体的血细胞线粒体DNA进行突变分析。结果 发现1例患者线粒体DNA 12S rRNA基因1438位点存在G→A的点突变,另1例存在1442位点G→A的点突变,所有对照个体均未发现该两位点的突变。未发现线粒体基因12S rRNA 1310位点C→T点突变。结论 1438位点G→A、1442位点G→A的突变可能与2型糖尿病的发生相关,该两位点突变的具体意义如何尚需进一步研究。1310位点C→T在血细胞中的突变率可能较低,进一步说明2型糖尿病的发生在线粒体遗传上具有一定的异质性。     

江苏宿迁地区Hp感染及粪便基因检测克拉霉素耐药现状

目的 了解江苏宿迁地区幽门螺杆菌（Hp）感染及对克拉霉素的耐药情况。方法 收集2016年10月~2017年10月在宿迁市第一人民医院行14C呼气试验检查的健康体检者的Hp检查结果,随机留取412例受检者的个人资料,并随机留取146例Hp感染者的粪便标本行粪便基因克拉霉素药敏试验,统计Hp感染率,比较相关因素对Hp感染的影响,初步了解Hp克拉霉素耐药情况。结果 共2919例纳入研究,1248例Hp阳性（42.75%）,男性人群的Hp感染率为44.89%,高于女性的39.89%（P＜0.05）,20岁以     

幽门螺杆菌对克拉霉素耐药分子机制研究进展

幽门螺杆菌（Hpylori,Hp）是慢性胃炎,消化性溃疡的重要致病因素。研究表明,Hp的根治和清除有利于溃疡的治愈。而Hp耐药直接影响Hp的根治和清除,Hp耐大环内酯类药物（克拉霉素）的机制与其23SrRNA的V区上的点突变有关。随着Hp耐药率的逐年升高,有关Hp的耐药分子机制和检测技术成为研究热点。因此,开展Hp耐药的相关研究对Hp的诊断和治疗有重大意义。     

4023例新生儿耳聋基因线粒体12S rRNA突变的研究

目的本研究通过对新生儿耳聋基因线粒体12S rRNA突变的检测,为预防药物性耳聋的可行性提供支持。方法对4023例新生儿采用飞行时间质谱检测技术进行耳聋基因线粒体12S rRNA的检测,检测位点包括A1555G和C1494T。结果4023例新生儿耳聋基因检测结果中共发现5例12S rRNA A1555G纯合突变,突变比例为0．12％。结论耳聋基因线粒体12SrRNA的检测对预防药物性耳聋具有重大意义,倡导对新生儿进行耳聋基因线粒体12S rRNA筛查的理念。     

利用寡核苷酸芯片检测乙型肝炎病毒基因突变

目的　利用寡核苷酸芯片平行分析乙肝病毒表面抗原区S、前核心抗原pre C区、X区、聚合酶P区 12个已知的突变位点。方法　针对S、pre C、X、P区的 12个突变位点 ,设计位于乙肝病毒反义链的 2 4条寡核苷酸探针和两对PCR引物 ,探针长度为 14～ 18bp ,其 5′端连有氨基己烷和T15间隔子 ,合成后经点样仪点到醛基化玻片上。两对PCR引物分别用于扩增S与P区内的 5个突变位点和X、前C区内的 7个突变位点 ,其中上游引物含有荧光标记。不对称PCR扩增所得到的荧光标记的单链DNA与寡核苷酸阵列杂交、清洗后经扫描分析实验结果。结果　在对 12个乙肝阳性样品前核心抗原C和X区的突变检测中 ,存在 176 2A→T ,176 4G→A联合突变的有 2例、1896G→A突变的 3例、同时存在 176 2A→T、176 4G→A联合突变和 1896G→A突变的 1例、未存在任何突变的样品 6例。在 12个乙肝阳性样品的表面抗原S的突变检测中 ,未发现有突变出现。部分样品的随机测序结果与寡核苷酸阵列杂交结果一致。结论　寡核苷酸芯片适于快速、平行、大量检测突变     

氨基糖甙类抗生素耳毒性相关的线粒体DNA突变

线粒体12S rRNA基因是引起氨基糖甙类抗生素耳毒性和非综合征型耳聋的突变热点区域。其中,位于高度保守的12S rRNA基因解码区的同质性1555A＞G和1494C＞T突变,可导致部分患者对氨基糖甙类抗生素超敏感。当发生1555A＞G或1494C＞T突变时,12S rRNA高度保守的A位就会形成新的1494C-G1555或1494U-A1555碱基配对,使得人类线粒体核糖体的结构与细菌核糖体更加相似,以致氨基糖甙类抗生素与12S rRNA的结合更加容易,从而解释了为何携带这些突变的个体在使用了氨基糖甙类抗生素后会出现或加重耳聋表型。相关功能研究表明,无论是否存在氨基糖甙类抗生素的作用,携带1555A＞G或1494C＞T突变的细胞均会出现线粒体蛋白合成缺陷,并随之引发细胞呼吸功能障碍。此外,携带这些突变的家系,其母系成员听力损失程度、发病年龄和外显率均存在较大差异,提示核修饰基因、线粒体单体型以及氨基糖甙类抗生素等对12S rRNA 1555A＞G和1494C＞T突变的表型表达起着修饰作用。这些研究成果为以下三个方面提供了科学依据：①预测个体耳毒性风险;②提高氨基糖甙类抗生素治疗的安全性;③降低耳聋发生率。     

应用扩增受阻突变检测系统（ARMS）检测HBV基因型

目的建立扩增受阻突变检测系统（ARMS）检测乙型肝炎病毒（HBV）基因型方法,并对慢性乙型肝炎（CHB）患者HBV基因型进行分析。方法通过对HBV全基因组序列比对分析,设计ARMS特异性引物和探针体系,建立检测HBVB和C基因型方法,对50例临床标本进行检测。结果50例临床样本中,B型的检出率为26％（13例）,C型为74％（37例）,其实验结果与测序结果完全一致。常州及周边地区乙型肝炎病毒C基因型HBV为优势株。C基因型患者A1762T／G1764A突变率为62％,明显高于B型。结论ARMS检测HBV基因型,快速、准确;常州及周边地区HBV基因型主要为B、C型,且C型携带A1762T／G1764A双突变率较高,与较为严重的肝病相关。     

母系遗传耳聋大家系的线粒体基因组全序列分析

目的 在江苏淮阴一母系遗传非综合征型耳聋大家系中，寻找线粒体基因组上可能影响1555（A→G）突变表型的其他位点突变。方法 采用聚合酶链反应-限制性片段长度多态性分析（PCR-restriction fragment length polymorphism，PCR-RFLP）和测序技术。检测了核心分支家系中27名母系成员的线粒体DNA上1555位点和7445位点的碱基变化，进而对该家系2名母系成员的线粒体全基因组和其他25名母系成员线粒体12S rRNA基因MTRNR1和tRNA-Ser^（UCN）基因MTTS1进行了全长测序。结果 再次证明了1555（A→G）突变是该家系成员致聋的分子生物学基础之一；并发现该家系27名母系成员的线粒体基因组中除1555（A→G）突变外，还同时存在有955-960（insC）同质型突变，两突变共分离。另外，新发现一个线粒体DNA突变——7449（insG），但该突变仅在2名母系成员中存在。结论 推测955-960（insC）突变可能通过改变12S rRNA基因的高级结构，并与1555（A→G）突变协同作用，提高了突变携带者对氨基糖甙类药物的敏感性；同时该突变可能也会导致线粒体蛋白质的合成缺陷。从而提高1555（A→G）突变致聋的外显率。     

Mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance

Among 12 clarithromycin-resistant Helicobacter pylori strains isolated in Guri, Korea, 8 showed an adenine to guanine mutation at position 2143 (formerly A2144G or E. coli 2059) in the 23S rRNA gene by the PCR-restriction fragment length polymorphism (RFLP) method. The remaining 4 strains, digested by neither BsaI nor BbsI, showed a thymine to cytosine mutation at position 2182 (T2182C) by direct sequencing of the PCR products. The T2182C mutants showed a tendency of higher levels of minimum inhibitory concentration to clarithromycin than the A2143G mutants. In conclusion, either the A2143G or the T2182C mutation was present in 100% of clarithromycin-resistant H. pylori isolates examined. The PCR-RFLP technique with restriction enzymes BbsI and BsaI was a rapid and relatively simple method to detect the clarithromycin resistance. But undigested isolates were quite frequent among our isolates (33.3%), the PCR-RFLP method with restriction enzymes BbsI and BsaI should not be used alone, and development of other rapid detection method for clarithromycin resistance is mandatory.     

Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals.

We designed a polymerase chain reaction (PCR) for amplifying the Helicobacter pylori gene encoding 16S rRNA. Primers for the specific detection of H. pylori were designed for areas of the 16S rRNA gene in which there is the least sequence homology between H. pylori and its closest relatives. The specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from the campylobacters H. cinaedi, H. mustelae, and Wolinella succinogenes, which are the closest relatives of H. pylori, as determined by 16S rRNA sequencing. Serial dilution experiments revealed the detection of as little as 0.1 pg of DNA by PCR and 0.01 pg by nested PCR. H. pylori DNA was detected successfully in clinical paraffin-embedded and fresh gastric biopsy specimens from patients positive for the bacterium and also in fecal suspensions seeded with the organism. The DNA from the nonculturable coccoid form of H. pylori was also identified by the primers. Universal primers designed for highly conserved areas on the 16S rRNA gene enabled large amplification products to be produced for direct sequencing analysis. Gastric bacteria resembling H. pylori have been isolated from animals. DNA of these animal gastric bacteria amplified with H. pylori-specific primers yielded PCR products identical to those from human isolates of H. pylori, as confirmed by the use of a 20-base radiolabelled probe complementary to an internal sequence flanked by the H. pylori-specific primers. The results of PCR amplification and partial 16S rRNA gene sequence analysis strongly support the contention that the gastric organisms previously recovered from a pig, a baboon, and rhesus monkeys are H. pylori.     

Real-time PCR for Helicobacter pylori quantification and detection of clarithromycin resistance in gastric tissue from patients with gastrointestinal disorders

Helicobacter pylori gram-negative bacteria commonly infect the human gastrointestinal (GI) tract and are readily diagnosed by endoscopy. H. pylori infection causes a broad range of host symptoms from discomfort to significant GI disorders (GIDs). Severity of the clinical manifestations depends mainly upon bacterial load. In this cross-sectional study, we investigated the affects of 23S rRNA point mutations on H. pylori count in naturally infected human GI tissues. Two-hundred H. pylori patients with suspected GIDs were evaluated to determine bacteria concentration and presence of four known 23S rRNA point mutations, causing clarithromycin resistance. Gastric biopsy specimens were examined by rapid urease test and 16S rRNA-targeted PCR to identify H. pylori; then bacterial load was quantified by real-time PCR targeting wild type and known 23S rRNA mutations. Eighty-two percent of the samples were confirmed as H. pylori-positive, having 10(4)-10(12) colony-forming units (CFU)/ml. The 10(6) load was most strongly associated with peptidyltransferase point mutations of the 23S rRNA gene A2144G (p = 0.033), A2143G (p = 0.005), A2143C (p = 0.005), and A2142G (p = 0.015). Thus, our findings indicated that dominant 23S rRNA mutated H. pylori strains have the same growth rate as the wild type in a gastric environment.     

Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens

A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing.     

Potential role of Helicobacter pylori in hepatocarcinogenesis

Helicobacter species can induce carcinoma in the liver of certain mice. Furthermore, Helicobacter pylori (H. pylori) exhibits hepatotoxicity in vitro. These reports indicate that H. pylori may play a role in hepatocarcinogenesis. The aim of this study was to assess the presence of H. pylori in human hepatocellular carcinoma (HCC) to determine if H. pylori may affect the development of this disease. Liver specimens from 15 HCC patients dissected into tumor and non-tumor tissues were examined for H. pylori by PCR using two sets of primers for 16S rRNA and urease B. DNA sequencing analysis was performed to confirm that PCR products with 16S rRNA primers were derived from H. pylori DNA. The specimens were also examined for H. pylori by immunohistochemistry using anti-H. pylori antibody. H. pylori was found in 13 of 15 tumor tissues, not in the non-tumor tissues. By contrast, Escherichia coli and Bacteroides fragilis, frequent colonizers of gut, were not detected by PCR in the HCC tumors. Ten cirrhotic liver tissue specimens and seven normal liver tissue specimens were also negative for H. pylori DNA by PCR. The nucleotide sequence of the amplified fragment shared 100% identity with the 16S rRNA gene of H. pylori. H. pylori was also detected in HCC tissue by immunohistochemical analysis. The presence of H. pylori in human HCC tissue was demonstrated by PCR and immunohistochemical analysis. These findings suggest that H. pylori might contribute to the development of HCC. Further study is needed to prove the pathogenetic role of H. pylori in the development of human HCC.     

Formation of A2143G mutation of 23S rRNA in progression of clarithromycin resistance in Helicobacter pylori 26695

The aim of this work was to investigate the link between 23S rRNA mutation and clarithromycin (CLR)-resistant Helicobacter pylori (H. pylori). CLR-resistant (CLRr) H. pylori strains were selected from the parent strain, H. pylori 26695, using medium containing CLR. Point mutations of 23S rRNA were analyzed by denaturing high-performance liquid chromatography and sequencing and restriction fragment length polymorphism (RFLP). Protein profiles of these strains were obtained by surface-enhanced laser/desorption ionization time-of-flight mass spectrometry technology. Two phenotype-stable L-CLRr resistant strains (MIC 8, 10 microg/ml) and two subsequent CLRr strains (MIC 32 microg/ml) were obtained. An A2143G mutation of 23S rRNA was detectable in two CLRr strains, but in neither the CLRs parent strain nor the L-CLRr strains. Four clinical CLRr H. pylori strains were obtained from 41 Chinese H. pylori isolates. The A2143G mutation was observed in all four CLRr isolates, but not in the six analyzed CLRs ones. Protein profiling showed that the pattern of protein expression was changed from the CLRs parent strain to the L-CLRr strains and then to the CLRr strains progressively. The formation of A2143G mutations of 23S rRNA might be a late event in the development of CLR-resistance of H. pylori 26695. Early events related to alteration of the pattern of protein expression might be involved in the development of CLR-resistance too.     

Identification of a mutation associated with erythromycin resistance in Bordetella pertussis: implications for surveillance of antimicrobial resistance

Erythromycin treatment failures and in vitro resistance of Bordetella pertussis have been reported on several occasions in the past few years, but the mechanism of resistance has not been described. One potential mechanism, genetic modification of the erythromycin-binding site on the 23S rRNA of the 50S ribosomal subunit, has been observed in other bacteria. To explore this possibility, we amplified the portion of the 23S rRNA gene encoding the central loop of domain V. DNA sequencing and restriction fragment length polymorphism of the PCR products showed that each of the four erythromycin-resistant B. pertussis strains tested contained an A-to-G transition mutation at position 2058 (Escherichia coli numbering) of the 23S rRNA gene. The mutation was not found in seven erythromycin-susceptible isolates tested. Two of the resistant isolates were heterozygous, containing at least one mutant copy and one wild-type copy of the 23S rRNA gene. These results indicate that erythromycin resistance in these strains is likely due to a mutation of the erythromycin-binding site in the 23S rRNA gene. Identification of the resistance mechanism will facilitate development of molecular susceptibility testing methods that can be used directly on clinical specimens in the absence of an isolate.     

Quadruplex real-time PCR assay using allele-specific scorpion primers for detection of mutations conferring clarithromycin resistance to Helicobacter pylori

We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori--the wild-type sequence and the three mutations conferring clarithromycin resistance--using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when the probes are annealed, are read in four channels by a SmartCycler thermocycler. The assay was first applied successfully on 4 reference and 61 clinical strains. MICs of clarithromycin were determined by the Etest method. A perfect concordance was obtained between Etest and Scorpion PCR. Mixed populations were better detected by Scorpion PCR. We examined 259 biopsies from 229 patients by culture, PCR-restriction fragment length polymorphism (RFLP), and Scorpion PCR. One biopsy, positive for culture, exhibited inhibitors for both PCR-RFLP and Scorpion PCR. Twelve biopsies were positive for PCR-RFLP and Scorpion PCR but negative for culture with concordant determination of mutations in the 23S rRNA genes by the two PCR assays. Three biopsies were positive for Scorpion PCR only. Compared to culture, the sensitivity of Scorpion PCR was 98.3% and the specificity was 92.5%. The Scorpion PCR assay provides a highly accurate, rapid, and precise method for the detection and determination of mutations conferring clarithromycin resistance to H. pylori.     

Detection of Helicobacter pylori DNA in peripheral blood from patients with peptic ulcer or gastritis

Cases of Helicobacter bacteremia have been reported from time to time. Helicobacter pylori is the most important representative of Helicobacterium, yet whether it can result in bacteremia has rarely been studied. In this study, we examined H. pylori DNA in peripheral blood and gastric mucosa of patients with peptic ulcer or chronic gastritis by polymerase chain reaction (PCR). We found H. pylori DNA in 15 of 20 gastric samples, and 9 of these specimens were positive for H. pylori culture. H. pylori DNA amplified by PCR was positive in the peripheral blood of three patients, who all had duodenal ulcers. Gastric biopsy specimens from these three patients were all positive for H. pylori genes and H. pylori was isolated from these specimens. After the 16S rRNA gene sequences of three specimens from the same patient were obtained, we found that they were identical, which suggested that they are the same strain. Our findings suggest that H. pylori exists not only in gastric mucosa but also in peripheral blood, and it is possible that H. pylori can result in bacteremia.