双重实时荧光PCR快速鉴别人参和西洋参＊

目的 建立一种能够快速鉴定人参和西洋参的双重实时荧光PCR方法。方法 通过对人参属及其近似种基因序列的分析比对，设计特异性的引物探针，建立双重实时荧光PCR检测方法。PCR反应体系为Premix Ex Taq （Probe qPCR） 10 μL，人参上下游引物各0.5 μL，西洋参上下游引物各0.3 μL，人参与西洋参特异性探针各0.4 μL，DNA模板2 μL，灭菌去离子水补至20 μL。反应条件为95℃预变性30 s；然后以95℃变性5 s，60℃退火延伸30 s进行40个循环。应用该方法测试了27份DNA样品，包括7份人参、6份西洋参、4份人参与西洋参混合样、10份其他人参属样品及其他常见中药材样品的DNA，以确定该方法的特异性。将人参与西洋参样品DNA混合后10倍稀释成不同浓度后进行检测，用以确定该方法的灵敏度。结果 人参及西洋参样品均在特定的荧光通道下出现典型的阳性扩增曲线，人参与西洋参混合样品均同时出现两条阳性扩增曲线，其它对照样品及空白对照均没有出现阳性扩增曲线。灵敏度检测结果显示该方法检测人参及西洋参的最低检测限均为2 pg DNA/反应。结论 本实验建立的双重实时荧光PCR方法能够同时快速、准确、灵敏地鉴别出人参和西洋参。     

微滴式数字PCR和实时荧光定量PCR对新型冠状病毒肺炎患者不同时间血便尿中核酸检测结果初步探讨

目的 分析微滴式数字PCR(droplet digital PCR, ddPCR)和实时荧光定量PCR(quantitative real-time PCR,qPCR)的核酸检测结果,比较两种方法检测各类样本的差异性,为改进新型冠状病毒核酸检测方案提供数据支持。 方法 利用ddPCR和qPCR技术对已经确诊的3例新型冠状病毒肺炎患者发病不同时间的全血、尿液、粪便共22份标本进行新型冠状病毒核酸检测。 结果 两种方法对人保守区域基因扩增结果一致:全血标本信号最强,尿液次之,粪便最少;ddPCR在1份全血,1份尿液,5份粪便中检出ORF-1ab和N基因的阳性微滴,qPCR仅在3份粪便中检出上述基因,漏检的3个标本基因拷贝数平均浓度为128 copies/ml;ddPCR在发病<5、5~15、>15 d的各类标本中都有检出,qPCR检出以中晚期为主;重症病例用ddPCR均可测到阳性微滴,qPCR检测的各类标本均为阴性;轻症病例的各类标本中qPCR只有粪便核酸检测阳性,ddPCR检出率高于qPCR。 结论 ddPCR可以有效克服qPCR 灵敏度不足的难题,是对qPCR 的有益补充,尤其是针对病毒载量比较低的血液、尿液和可疑的粪便或肛拭子标本,适用于早期感染的判断及患者治愈后出院诊断。     

EBUS-TBNA超声内镜对纵隔肿大淋巴结良恶性的鉴别诊断价值

目的:分析超声内镜引导下经支气管针吸活检(endobronchial ultrasound-guided transbronchial needle aspiration，EBUS-TBNA)术中肿大纵隔淋巴结的超声图像特点，探讨EBUS-TBNA超声内镜对纵隔淋巴结良恶性的鉴别诊断价值，以期提高EBUS-TBNA对纵隔淋巴结恶性病变的活检率。方法:回顾性分析2014年10月至2018年11月行EBUS-TBNA患者的超声内镜图像。我们使用以下EBUS超声内镜特征来预测淋巴结的良恶:回声，长轴长度，短轴长度，纵横比，形态，边界，淋巴门有无，淋巴结内血流信号分级。将超声检查结果与最终病理结果或临床随访结果进行比较。采用SPSS 20.0软件进行统计学分析，采用logistic回归分析评价肿大淋巴结EBUS-TBNA超声内镜下的特征与良恶性的相关性，以 P<0.05 为标准判定差异有统计学意义。结果:对130例纵隔淋巴结肿大患者的227个淋巴结进行回顾性分析，67.4%的肿大淋巴结被证实为恶性转移。Logistic回归分析显示回声、长轴及短轴的长度、正常淋巴门结构的消失是诊断恶性淋巴结的独立预测因素。结论:纵隔恶性淋巴结具有一定的超声特征，可以通过这些超声特征提高EBUS-TBNA对纵隔恶性淋巴结的检出率。     

PCR仪与测序仪应用于丙型肝炎病毒基因型检测的临床效果观察

目的:观察PCR仪与测序仪在丙型肝炎病毒(HCV)基因型检测中的应用。方法:选择2018年1月~2018年12月收治的134例HCV感染患者为研究对象,给予患者PCR仪检测、测序仪检测。结果:测序仪检测134例均可分型,PCR仪检测130例患者可分型,4例未能分型。PCR检测结果:134例HCV感染患者中,1b型59例,2a型47例,3a型6例,3b型15例,6a型3例,4例为分出型别。基因测序结果:134例均可分型:1型59例,2a型40例,2i型7例,3a型6例,3b型15例,6a型3例,6n型4例。PCR仪检测法检测符合率97.0%。结论:PCR仪测序仪检测用于丙型肝炎病毒基因型检测,操作便捷,但其只能检测探针覆盖的型别,个别罕见型别不能分型。     

Genetic and epigenetic alterations of the EGFR and mutually independent association with BRCA1, MGMT,and RASSF1A methylations in Vietnamese lung adenocarcinomas

Genetic and epigenetic alterations importantly contribute to the pathogenesis of lung cancer. In the study, we measured the frequency and distribution of molecular abnormalities of EGFR as well as the aberrant promoter methylations of BRCA1, MGMT, MLH1, and RASSF1A in Vietnamese lung adenocarcinomas. We investigated the association between genetic and epigenetic alteration, and between each abnormality with clinicopathologic parameters. Somatic EGFR mutation that was found in 49/139 (35.3%) lung adenocarcinomas showed a significant association with young age, female gender, and non-smokers. EGFR overexpression was identified in 82 tumors (59.0%) and statistical relationships with EGFR or BRCA1 methylation but not EGFR mutation. In addition, EGFR, BRCA1, MGMT, MLH1, and RASSF1A methylations were found in 33 (23.7%), 41 (29.5%), 46 (33.1%), 28 (20.1%), and 41 (29.5%) cases of a total of 139 lung adenocarcinomas, respectively. The RASSF1A methylation was found to be linked to the smoking habit. Methylations in MGMT and RASSF1A were also found to correlate with metastasis status. Furthermore, the distribution of EGFR mutation and that of BRCA1, MGMT or RASSF1A methylation were significantly exclusive in lung adenocarcinomas. The main finding of our study demonstrate that epigenetic abnormalities might play a critical role for the lung tumorigenesis in patients with smoking history and metastasis, and partly affect the predictive value of EGFR mutations through blocking expression due to promoter EGFR hypermethylation. Mutually exclusive distribution of genetic and epigenetic alterations reflects differently biological characteristics in the etiology of lung adenocarcinomas.     

ONRAB® oral rabies vaccine is shed from,but does not persist in,captive mammals

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.     

Spread of an Enterococcus faecalis sequence type 6 (CC2) clone in patients undergoing selective decontamination of the digestive tract

Enterococcus faecalis (E. faecalis) is a common cause of nosocomial infection in immunocompromised patients. The presence and dissemination of high‐risk clonal complexes, such as CC2, is an ongoing problem in hospitals. The aim of this work was to characterize 24 E. faecalis isolates from ICU patients undergoing selective decontamination of the digestive tract (SDD) by phenotypical (antimicrobial susceptibility) and genotypical (presence of virulence genes, RAPD‐PCR and MLST) methods. Our results showed high prevalence of the ST6 E. faecalis clone (91.6%), especially adapted to the hospital environment, with a multidrug resistance pattern and a multitude of putative virulence genes. In addition, ST179 (4.2%) and ST191 (4.2%) were detected. By RAPD–PCR analysis, the 22 isolates identified as ST6 showed six different DNA patterns, while the two remaining isolates, ST179 and ST191, showed two additional profiles. CC2 is a known clonal complex with high adaptability to hospital environment and worldwide distribution. The high prevalence of the ST6 clone in the studied population could be related to the presence of gentamicin in the SDD mixture since most strains were gentamicin resistant. Consequently, strict surveillance should be applied for rapid detection and control of this clone to prevent future spread outside the ICU.     

基于荧光定量PCR技术的当归不同炮制品中大肠埃希菌数量测定方法开发及比较研究

目的对当归不同炮制品中大肠埃希菌变化进行定量分析。方法建立荧光定量PCR方法,对当归不同炮制品、不同产地、不同收集企业、不同储藏时间的大肠埃希菌进行定量分析。结果不同炮制品中大肠埃希菌数量变化为生当归土炒当归酒当归;在不同产地的当归中以甘肃岷县地区所含的大肠埃希菌的数量最少;与零售企业相比,生产销售企业的当归和酒当归中大肠埃希菌的数量较少;不同储藏时间对当归和酒当归中大肠埃希菌数量有一定影响,随储藏时间的增加,大肠埃希菌的数量也在增加。对部分代表性样品进行平板计数法与荧光定量PCR法比较时发现,平板计数法结果多呈阴性,荧光定量PCR结果均呈阳性。结论所建立的荧光定量PCR法特异性、灵敏度、可靠性以及报告周期均优于平板计数法,可为当归不同炮制品中大肠埃希菌的快速、准确定量检测提供有效手段。     

Evaluation of <i>MGMT</i> Gene Methylation in Neuroendocrine Neoplasms

Unresectable neuroendocrine neoplasms (NENs) often poorly respond to standard therapeutic approaches. Alkylating agents, in particular temozolomide, commonly used to treat high-grade brain tumors including glioblastomas, have recently been tested in advanced or metastatic NENs, where they showed promising response rates. In glioblastomas, prediction of response to temozolomide is based on the assessment of the methylation status of the MGMT gene, as its product, O6 -methylguanine-DNA methyltransferase, may counteract the damaging effects of the alkylating agent. However, in NENs, such a biomarker has not been validated yet. Thus, we have investigated MGMT methylation in 42 NENs of different grades and from various sites of origin by two different approaches: in contrast to methylation-specific PCR (MSP), which is commonly used in glioblastoma management, amplicon bisulfite sequencing (ABS) is based on high-resolution, next-generation sequencing and interrogates several additional CpG sites compared to those covered by MSP. Overall, we found MGMT methylation in 74% (31/42) of the NENs investigated. A higher methylation degree was observed in welldifferentiated tumors and in tumors originating in the gastrointestinal tract. Comparing MSP and ABS results, we demonstrate that the region analyzed by the MSP test is sufficiently informative of the MGMT methylation status in NENs, suggesting that this predictive parameter could routinely be interrogated also in NENs.