病原宏基因组高通量测序性能确认方案

宏基因组学利用新一代高通量测序技术, 以特定环境下病原体基因组为研究对象, 在分析病原体多样性、种群结构、进化关系的基础上, 进一步探究病原体的群体功能活性、相互作用及其与环境之间的关系, 发掘潜在的生物学意义。目前, 绝大部分的宏基因组学研究都集中在临床价值评价, 宏基因组检测临床应用前分析性能确认的研究相对空白, 北京协和医院检验科研究团队结合多年病原宏基因组检测的经验和国内外相关研究成果, 就病原宏基因组项目医院本地化开展前的性能确认工作, 从临床预期用途、方法学建立、性能确认、标准操作作业书4个方面提出了具体的实施方案, 具体包含:标本类型和病原体范围、生物信息流程建立、生物信息分析参考盘制备和评估、湿实验流程的建立、背景核酸数据模型的建立、参考盘制备和全流程的性能确认等。     

高通量测序在中草药研究中的应用

高通量测序技术,也被称为“下一代”测序技术,它能够一次对几十万到数百万条分子同时进行测序,是对传统测序的一次革命性改变,具有里程碑式的意义［1］。高通量测序经过数年的发展,已经成为一项较为成熟的研究手段,在转录组测序,基因甲基化,宏基因测序,基因组拼接,micro‐RNA 测序等方面极大地促进了中草药基因的研究发展,对传统中草药进行高通量测序研究,可以从整体水平上了解目标物种的功能基因概况,明确活性成分的代谢通路,为中草药研究奠定分子生物学基础,为传统中草药理论提供现代生物学阐释。本文对高通量测序技术在中草药研究中的应用综述如下。     

基于宏基因组二代测序诊断的鹦鹉热衣原体肺炎的流行病学特征分析

目的 基于宏基因组二代测序(mNGS)诊断的鹦鹉热衣原体肺炎的相关数据,分析其流行病学特征.方法 由微远基因科技有限公司和迪飞医学科技有限公司提供宏基因组二代测序数据,分别为数据集Ⅰ(2019年11月至2021年1月)和数据集Ⅱ(2019年4月至2020年12月),包括:鹦鹉热衣原体检测阳性患者的性别、年龄、所在城市、...     

新型宏基因组/宏转录组学技术在反向病原学研究中的应用

以微生物组学和宏基因组学为基础的新技术的飞跃式发展为新微生物的发现提供了无可比拟的优势。反向病原学理念公开提出后，在指导新发突发传染病的主动防控实践方面也正在发挥越来越多的作用，在公共卫生和疾病防控领域应用越来越广。新型宏基因组/宏转录组学技术进一步支撑和促进反向病原学研究，在发展评估新微生物致病潜力的理论、策略和方法方面有了一定的进展。在此，本综述梳理了新型宏基因组/宏转录组学技术在反向病原学研究中的作用实践、以及面临的挑战，期望促进人们对病原体的认识和理论技术创新。     

组学技术在布鲁菌病诊断中的应用研究进展

布鲁菌病是一种严重的人畜共患病,其诊断一直是个难题。随着组学研究和技术的不断发展,越来越多的学者开始关注布鲁菌病的组学研究。在基因组学方面,利用测序技术获得基因组数据,可以更好地了解菌株之间的遗传差异和相似性,从而对菌株进行分型,宏基因测序已经应用于布鲁菌病的诊断。在转录组学方面,通过测序技术获取病原体在特定条件下的转录组数据,寻找对诊断布鲁菌病有价值的生物标志物。在蛋白质组学方面,通过质谱技术分析蛋白质在生物学过程中的表达、互作和调控,为开发布鲁菌病的新型诊断方法提供支持。在代谢组学方面,利用液相色谱串联质谱技术获得代谢物的代谢谱,基于代谢组数据可以鉴定代谢途径和代谢产物,更好地了解布鲁菌菌株之间的代谢差异,为布鲁菌病的诊断提供了新的方向。多组学联合可以构建出更加全面而准确的生物学信息,为布鲁菌病的诊断提供新的思路和方法。     

病毒宏基因组在医学上的应用与未来展望

新型新冠病毒的爆发,给人类安全健康带来了巨大的威胁。在过去的经验中,发现和鉴定新病毒以及确定新病毒与疾病的关系是预防、诊断和治疗新发病毒性传染病的首要任务。在过去十几年中,随着高通量测序技术的迅速发展,病毒宏基因组的理论以及技术已被证明在公共卫生,临床病原学诊断等方面发挥着重要的作用。此前,人们对病毒的认识被限制于病毒感染人体进而引起疾病,但是近些年随着人类基因组和微生物组研究表明,病毒与人体之间存在更广泛的相互作用,部分病毒对人体无害,甚至是有益的。本文主要针对近些年来对病毒宏基因组的兴起与发展,研究的策略以及在医学上的一些应用前景进行综述,并提出病毒宏基因组的一些未来展望。     

IDseq&IDseq Ultra宏基因组高通量测序(mNGS)在感染性疾病诊断中的临床应用征文通知北大核心CSCD

宏基因组高通量测序(mNGS)能够快速、客观地检测临床样本中的病原微生物(包括细菌、真菌、病毒、寄生虫、非典型病原体等),主要应用于呼吸系统、中枢神经系统和循环系统感染性疾病中的诊断与鉴别诊断。为进一步提升临床感染性疾病的快速诊断能力,提高mNGS在感染性疾病中的有效应用及诊疗效益,中华急诊医学杂志社联合广州微远基因科技有限公司于2022年5月1日至2022年12月30日举办IDseq&IDSeq Ultra宏基因组高通量测序(mNGS)在感染性疾病诊断中的临床应用征文活动,现将具体事项通知如下。     

病原宏基因组测序技术及其在临床感染辅助诊断中的应用研究

近年来,病原宏基因组二代测序（metagenomic next-generation sequencing,mNGS）技术在临床感染领域的应用越来越广泛。在临床需求的推动下,mNGS技术也在不断地优化和发展。从手工操作到自动化流程,从单纯定性检测到定量监控,从感染鉴别到“感染+肿瘤”多维诊断都取得了一定的研究成果。当然,该技术在临床应用中还存在一定的局限性,如目前国内mNGS检测相关试剂众多,但尚未建立系统完整的质量管理控制和评价体系,尚需进一步研究。该文就近年来病原宏基因组测序技术的发展、应用以及mNGS检测的标准化和规范化流程进行了总结,以期为病原mNGS测序更好地辅助临床感染诊断提供参考。     

16S rDNA测序技术在婴儿肠道微生态研究中的应用

目的探讨16SrDNA测序技术在新生儿、婴儿肠道微生态研究中的应用。方法于生后3天、1月、6月、1岁时收集2例健康婴儿粪便标本共8份,提取细菌总DNA,以Illumina Hiseq 2000为测序平台,采用新一代高通量16SrDNA宏基因组测序技术对V6可变区测序,并进行生物信息分析(物种分类和丰度分析;多样性分析)。结果 8份样品共产生原始测序数据为1 027.47 Mbp,Unique tags序列数量均值为58630,OTU数量63~209;优势菌门为Proteobacteria和Firmicutes;在科水平,1%的物种1个月之内2~4种,6月后达7~10种;1号婴儿一直以Enterobacteriaceae占优势,2号婴儿优势菌群包括Enterobacteriaceae、Lachnospiraceae、Streptococcaceae和Bacteroidaceae;4个时间点的npShannon和Simpson指数分别为1.17、1.29、2.16、2.51和0.43、0.40、0.26、0.14。结论 16SrDNA测序技术能满足新生儿、婴儿肠道微生态研究需求;新生儿、婴儿粪便中含丰富细菌基因组;细菌物种丰度及分类存在个体差异;从出生到1岁,婴儿肠道菌群结构趋向复杂和多样。     

我国西北地区蚊虫标本中广平病毒的检测

目的 对我国西北地区蚊虫携带病毒开展调查,并快速发现重要的蚊媒病毒.方法 通过形态学方法对采集蚊虫进行分类鉴定,深度测序和宏基因组分析方法分析蚊虫携带的病毒,结合Sanger测序法补全病毒基因组全序,应用系统进化分析方法分析病毒分子遗传特征.结果 2019年7月采集自陕西省的中华按蚊标本和宁夏回族自治区的三带喙库蚊标本...     

Identification of Bacillus anthracis by multiprobe microarray hybridization

We have developed a rapid assay based on microarray analysis of amplified genetic markers for reliable identification of Bacillus anthracis and its discrimination from other closely related bacterial species of the Bacillus cereus group. By combining polymerase chain reaction (PCR) amplification of six B. anthracis-specific genes (plasmid-associated genes encoding virulence factors (cyaA, pagA, lef, and capA, capB, capC) and one chromosomal marker BA-5449) with analysis of amplicons by microarray hybridization, we were able to unambiguously identify and discriminate B. anthracis among other closely related species. Bacillus identification relied on hybridization with multiple individual microarray oligonucleotide probes (oligoprobes) specific to each target B. anthracis gene. Evaluation of the assay was conducted using several B. anthracis strains (with or without pXO1 and pXO2 plasmids) as well as over 50 other species phylogenetically related to B. anthracis, including B. cereus, B. thuringiensis, B. mycoides, and B. subtilis. The developed microarray analysis of amplified genetic markers protocol provides an efficient method for (i) unambiguous identification and discrimination of B. anthracis from other Bacillus species and (ii) distinguishing between plasmid-containing and plasmid-free Bacillus anthracis strains.     

尿培养产CTX-M大肠埃希菌的种系分型及耐药和毒力特点分析

目的分析尿培养产CTX-M大肠埃希菌的种系分型,耐药及毒力特点。方法收集该院2014年尿培养大肠埃希菌,环扩散法测定细菌的敏感性,ESBLs确定分析细菌产ESBLs的情况;采用肠杆菌属重复基因间隔共有序列-PCR(ERIC-PCR)对细菌进行遗传相关性分析;PCR扩增CTX-M编码基因和毒力基因iutA,ompT,fyuA,fdeC,fimH,traT,cvaC,pap,kpsMT,pAI,usp,aer,hlyA,cnf,和chuA;多重PCR分析产CTX-M大肠埃希菌的种系分型;依据PCR对菌株的CTX-M编码基因的检验结果,将细菌分为产CTX-M组和非产CTX-M组,对比分析两组之间在抗菌药物耐药性和毒力基因之间的差异。结果 162株尿培养大肠埃希菌中没有遗传相关性,126株ESBLs阳性菌株中,有91株细菌产CTX-M,其中,57株产CTX-M大肠埃希菌属于D型,16株属于B2型。统计学分析发现,产CTX-M组细菌的耐药率显著高于不产CTX-M组细菌(除外亚胺培南),毒力基因iutA、chuA和traT在产CTX-M细菌组中的流行显著高于非产CTX-M组,P值分别依次为:0.001、0.006和0.000。结论产CTX-M大肠埃希菌是本院尿路感染的主要致病菌,大多属于D型,其耐药率显著增高,且与毒力基因iutA、chuA和traT密切相关,是尿路感染患者临床治疗的一个潜在威胁。     

Complementation of the exoS gene in the pvdE pyoverdine synthesis gene-deficient mutant of Pseudomonas aeruginosa results in recovery of the pvdE gene-mediated penetration through the intestinal epithelial cell barrier but not the pvdE-mediated virulence in silkworms

Translocation of endogenous Pseudomonas aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Comparative genome hybridization analysis of high virulent and low virulent strains allowed us to identify bacterial genes that are associated with bacterial translocation from gut in infected hosts. Here we focused on the pvdE pyoverdine synthesis gene among the identified bacterial genes, showing that the pvdE gene is required for bacterial penetration through epithelial cell monolayers and for bacterial translocation from gut to hemolymph in infected silkworms. We next revealed that mRNA expression level of the exoS gene in a pvdE-deficient mutant (ΔpvdE) after incubation with Caco-2 cells was greatly reduced as compared with that in the wild-type strain. The pvdE- and exoS-complemented ΔpvdE strains (ΔpvdE/pvdE and ΔpvdE/exoS) showed recovery of the ability of bacterial penetration through Caco-2 cell monolayers and of the ability of bacterial translocation from gut to hemolymph in infected silkworms. However, there were differences between the ability of ΔpvdE/pvdE and ΔpvdE/exoS to kill silkworms after intestinal infection and to replicate in hemolymph following direct injection into the hemolymph: ΔpvdE/pvdE could kill silkworms after intestinal infection and could replicate in hemolymph to levels similar to those of the wild-type strain, but ΔpvdE/exoS could not. Taken together, our results suggest that the virulence of the wild-strain mediated by the pvdE gene is the result of the ability to both penetrate through the intestinal epithelial cell barrier depending on ExoS and to replicate in hemolymph independently of ExoS.     

Multiple genetic elements carry the tetracycline resistance gene tet(W) in the animal pathogen Arcanobacterium pyogenes

The tet(W) gene is associated with tetracycline resistance in a wide range of bacterial species, including obligately anaerobic rumen bacteria and isolates from the human gut and oral mucosa. However, little is known about how this gene is disseminated and the types of genetic elements it is carried on. We examined tetracycline-resistant isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes, all of which carried tet(W), and identified three genetic elements designated ATE-1, ATE-2, and ATE-3. These elements were found in 25%, 35%, and 60% of tetracycline-resistant isolates, respectively, with some strains carrying both ATE-2 and ATE-3. ATE-1 shows characteristics of a mobilizable transposon, and the tet(W) genes from strains carrying this element can be transferred at low frequencies between A. pyogenes strains. ATE-2 has characteristics of a simple transposon, carrying only the resistance gene and a transposase, while in ATE-3, the tet(W) gene is associated with a streptomycin resistance gene that is 100% identical at the DNA level with the aadE gene from the Campylobacter jejuni plasmid pCG8245. Both ATE-2 and ATE-3 show evidence of being carried on larger genetic elements, but conjugation to other strains was not observed under the conditions tested. ATE-1 was preferentially associated with A. pyogenes strains of bovine origin, while ATE-2 and ATE-3 elements were primarily found in porcine isolates, suggesting that these elements may circulate in different environments. In addition, four alleles of the tet(W) gene, primarily associated with different elements, were detected among A. pyogenes isolates.     

VraSR two-component regulatory system contributes to mprF-mediated decreased susceptibility to daptomycin in in vivo-selected clinical strains of methicillin-resistant Staphylococcus aureus

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains.     

Chromosomally mediated beta-lactamase production and gentamicin resistance in Enterococcus faecalis.

We have analyzed four distinct strains of multiply resistant, beta-lactamase-producing enterococci isolated during an outbreak of colonization with these strains on an infant-toddler surgical ward at The Children's Hospital in Boston, Mass. All four strains were resistant to erythromycin, penicillin, and tetracycline and to high levels of gentamicin and streptomycin. One strain was also resistant to chloramphenicol. Plasmid profiles revealed four different plasmid patterns, with the number of identified plasmids ranging from zero to three. The gene coding for beta-lactamase production could be transferred at low frequency (less than 10(-8)) to an enterococcal recipient from one strain in conjunction with all of the other resistance determinants. Probes derived from the staphylococcal beta-lactamase gene and gentamicin resistance gene failed to hybridize with any of the detectable plasmids, but both genes were present on restriction fragments of genomic DNA in all strains. Our results indicate that the beta-lactamase genes and gentamicin resistance genes in these strains are integrated into the bacterial chromosome. The cotransmissibility of the resistance determinants raises the possibility of their incorporation into a multiresistance transposable genetic element.     

tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus

The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus. By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.     

Multifocal outbreaks of metallo-beta-lactamase-producing Pseudomonas aeruginosa resistant to broad-spectrum beta-lactams, including carbapenems.

A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994. Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the metallo-beta-lactamase gene (blaIMP)-specific probe. Fifteen strains carrying the metallo-beta-lactamase gene were identified in five hospitals in different geographical areas. Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between. These results revealed that the acquisition of a metallo-beta-lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the metallo-beta-lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital. These findings suggest that the metallo-beta-lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the metallo-beta-lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of metallo-beta-lactamase-producing strains with resistance to various beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of metallo-beta-lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum beta-lactams including carbapenems.