Establishment of Multiplex PCR for Simultaneous Detection of Four Venereal Pathogens

Venereal diseases are considered to be the most prevalent infectious diseases in the worldwide. China is now faced with a year-by-year increasing incidence of sexually transmitted diseases （STD）, which are spreading from high-risk groups to the general population. Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum and herpes simplex virus-2 （HSV-2） are always regarded as the most common venereal pathogens. The ＂golden standard＂ for testing Neisseria gonorrhoeae remains to be bacteria culture or microscopic examination.

Detection and Identification of Sugarcane Pokkah Boeng Pathogens of Main Sugarcane Varieties in Yunnan Province

[Objectives]This study was conducted to identify the pathogen species and dominant species of sugarcane pokkah boeng in main varieties in Yunnan sugarcane areas,so as to promote the healthy and sustainable development of sugarcane industry.[Methods]Specific primers Fv-F3/Fv-R3 and Fp-F4/Fp-R4 were designed based on the ribosomal DNA non-internal transcribed spacer(rDNA-ITS)gene sequences of Fusarium verticillioides and Fusarium proliferarum,the main pathogens of sugarcane pokkah boeng,and 117 typical sugarcane pokkah boeng samples collected from main varieties in different sugarcane areas of Yunnan Province were detected and analyzed by PCR.[Results]Among the 117 sugarcane pokkah boeng samples,112 samples were detected with F.verticillioides with a positive detection rate of 95.7%;103 samples were detected with F.proliferarum with a positive detection rate of 88%;103 samples were infected by F.verticillioides+F.proliferarum,and the compound infection rate was 88%;and the two pathogens were not detected in 5 samples,which might be sugarcane pokkah boeng caused by other species.PCR amplification products of 23 F.verticillioides positive samples and 19 F.proliferarum positive samples from different sugarcane varieties in different sugarcane areas were sequenced.The BLAST alignment results showed that the sequences of the 23 amplification products of F.verticillioides shared 99.45%-100.00%homology with F.verticillioides(GenBank accession number:KU508286),and the sequences of the 19 amplification products of F.proliferarum shared 99.26%-100.00%homology with the sequence of F.proliferarum(GenBank accession number:MK252904).Part of the F.verticillioids and 11 F.proliferarum sequences were selected to construct a phylogenetic tree,and the phylogenetic analysis showed that they belonged to the F.verticillioids group and F.proliferarum group,respectively.The results showed that F.verticillioides and F.proliferarum were the main pathogens causing sugarcane pokkah boeng of the main sugarcane varieties in Yunnan,and there was a common phenomenon of compound infection.F.verticillioides was the dominant species in Pu er,Lincang,Honghe and Yuxi sugarcane areas,but the detection rate of F.proliferarum was also high,and there were other species.In the future,the discovery of resistant germplasm resources and breeding of resistant varieties should be carried out aiming at these two pathogens of sugarcane pokkah boeng.[Conclusions]The study provides technical support for rapid identification of sugarcane pokkah boeng pathogens,and scientific basis for breeding resistant varieties and scientific disease prevention and control.

RVP Fast与Seeplex PneumoBacter ACE Detection assay联合诊断呼吸道感染病原体

目的采用RVP Fast与Seeplex PneumoBacter ACE Detection assay两种方法,联合诊断呼吸道感染病原体。方法收集南京市呼吸道感染患者咽拭子标本67份,提取RNA及DNA,呼吸道病毒群快速检测(Respiratory Virus Panel Fast Array,RVP Fast)技术检测呼吸道病毒,Seeplex PneumoBacter ACE Detection assay检测呼吸道六重病原体。结果 67份标本中,呼吸道病毒阳性仅4例,主要为肠鼻病毒。呼吸道六重病原体检测中,阳性标本为26例,其中肺炎链球菌、流感嗜血杆菌、肺炎衣原体阳性较多。结论 RVP Fast与Seeplex PneumoBacter ACE Detection assay联合使用,有利于明确呼吸道感染病原体。

A Study on Detecting and Identifying Enteric Pathogens With PCR

Objective To develop a rapid and definite diagnostic test of bacterial enteritis caused by pathogenic enterobacteria, the most frequent etiologic agent of infectious enteritis in the world. Methods A set of conventional PCR assays were applied to detect and identify salmonella, shigella, and E. coli O157:H7 directly from pure culture and fecal samples. The general primers of pathogenic enterobacteria were located on the uidA gene, which were found not only in E. coli nuclear acid, but also in Shigella and salmonella genes. Shigella primer was from ipaH gene whose coded invasive plasmid relative antigen existed both in plasmid and in genome. The primers of salmonella were designed from the 16SrRNA sequence. The primer of E.coli O157:H7 was taken from eaeA gene. Five random primers were selected for RAPD. The detection system included common PCR, semi-nested PCR and RAPD. Results This method was more sensitive, specific and efficient and its processing was rapid and simple. For example, the method could be used to specifically detect and identify salmonella, shigella, and E. coli O157:H7, and its sensitivity ranged from 3 to 50 CFU, and its detection time was 4 hours. Conclusion This PCR method, therefore, can serve as a routine and practical protocol for detecting and identifying pathogenic microorganisms from clinical samples.

Molecular detection of Xanthomonas oryzae pv.oryzae, Xanthomonas oryzae pv. oryzicola, and Burkholderia glumae in infected rice seeds and leaves

The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv.oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an Avr Rxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer(ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10 fg μL-1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.

Development of a Microfluidics-Based Quantitative Real-Time PCR to Rapidly Identify Photobacterium damselae subsp.damselae with Different Pathogenicity by Detecting the Presence of mcp or dly Gene

As a marine bacterial pathogen, Photobacterium damselae subsp. damselae(PDD) is distributed in seawater worldwide. It can infect different animals as well as humans, even cause deaths. The highly conserved regions of PDD mcp gene on chromosome and dly gene on plasmid were selected as the target fragments to design the specific primers. Recombinant plasmid standard was prepared based on the primers. With GENECHECKER UF-150 qRT-PCR instrument as the platform, a fluorescence-based quantitative real-time PCR(qRT-PCR) method was established for the detection of PDD. This method can specifically detect PDD and distinguish the highly virulent strains. Additionally, the test results can be qualitatively judged by visualization, while the quantitative detection can be achieved through the standard curve calculation. The minimum limit of detection was 1.0×101 copies μL-1, and the detection time was less than 20 min. In summary, this new method has outstanding advantages, such as strong specificity, high sensitivity, and low site requirements. It is a rapid on-site detection technology for highly virulent PDD strains.

Preliminary investigation and detection based on loop-mediated isothermal amplification(LAMP)of phytoplasmas associated with diseases in B.napus L.

In the last decade,some disease occurred on our experimental farms that had caused serious losses.They were not caused by fungi,bacteria or viruses.By loop-mediated isothermal amplification(LAMP)technique,the detection results pointed to the possible pathogen as phytoplasma.The investigation results implied that phytoplasmas could cause more than 13 kinds of symptoms in almost all parts of plants in B.napus L.,including witches’broom,multi-stems,aggregate main inflorescences,and flat stems.The incidences of these phytoplasma-associated diseases in our experimental farms rose from 1.61%in 2010 to 6.00%in 2021.Some phytoplasma infected plants died without any growing points.These studies would be helpful for detecting phytoplasmas diseases,selecting disease resistant germplasm and improving varieties with disease resistances in B.napus L.

Functional nucleic acid circuitry as a universal detector for pathogens

While the pathogen nucleic acid diagnostic technology has made tremendous progresses,several challenges,including long development cycles and limited specificity still exist,especially in the context of isothermal amplification techniques.To enhance the detection accuracy,here a functional strand displacement catalytic hairpin assembly circuit,which can perform at high-temperature(HT-CHA),was developed as the downstream of the loop mediated isothermal nucleic acid amplification(LAMP).The addition of HT-CHA not only ensures the specificity but also amplifies the detection signal.Taking African swine fever(ASF)gene as the target,the input of HT-CHA was designed according to the ASF gene LAMP amplicons.This LAMP-HTCHA can detect as low as 2 copies/μL targeting genes with high signal-to-noise ratio.Through importing a three-way junction(3WJ)transducer,the HT-CHA well-developed for ASF gene can be directly adapted to detect the LAMP amplicons of other pathogen genes,such as Mycoplasma pneumoniae(MP)gene.The time-consuming and high-risk process to redesign HT-CHA components can be further avoided,making the method even general and useful for a plenty of other targets.Finally,the accurate detection of MP gene in alveolar lavage fluid samples confirmed the high potential of the LAMP and HT-CHA combination in clinical applications,providing a promising strategy to develop point-of-care diagnostics at constant temperatures.

Distribution of Virulence-Associated Genes of Avian Pathogenic Escherichia coli Isolates in China

216 avian pathogenic Escherichia coli （APEC） isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as 078, 088, and 093. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD （70.8%）, iss （58.8%）, and tsh （51.4%） in APEC isolates. The detection rate of high pathogenicity islands （HPI）-associatedfyuA and irp2 genes were both 44.9%, with no LEE （the locus of enterocyte effacement） island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained onlyfimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% 078 isolates had a gene distribution patterns of fimC^＋iucD^＋irp2^＋fyuA^＋iss^＋colV^＋tsh^＋.

羊捻转血矛线虫病检测方法研究进展

羊捻转血矛线虫病是由捻转血矛线虫寄生于羊等小反刍动物皱胃内引起的一种危害严重的嗜血性寄生虫病。病羊临床表现为消瘦、贫血和衰弱等慢性消耗性症状,严重感染者可致死。该病在我国牧区普遍流行,目前暂无安全长效疫苗可用,加之耐药性严重,放牧羊群感染率和发病率居高不下,极大地降低了羊养殖业的经济效益。准确可靠的检测手段是诊断寄生虫感染、制定驱虫治疗方案、耐药监测及流行病学调查的关键环节。本文详细介绍了羊捻转血矛线虫病的检测技术研究现状,分析了病原学、免疫学及分子检测等方法的优缺点,并对未来检测技术发展趋势进行了展望,以期为该病新型检测方法的研发及综合防控策略提供参考。

4种食源性致病菌多重PCR检测体系的建立及其在乳品检测中的应用

食源性致病菌是通过食物进入人体引起食物中毒和诱发疾病的有害微生物,严重威胁着人类健康和环境安全,为了预防和控制食源性疾病的发生,有效对策之一是对食品中的病原微生物进行安全检测。本研究采用一种多重聚合酶链式反应法(multiplex polymerase chain reaction,MPCR)快速检测乳品中4种食源性致病菌,结果表明,选取单核细胞增生李斯特氏菌prfA基因、蜡样芽胞杆菌gyrB基因、鼠伤寒沙门氏菌invA基因、大肠埃希氏菌O157∶H7 stx2A基因的保守序列设计4对引物,在优化的PCR反应体系和退火温度58℃下,PCR扩增表现出良好的特异性,分别扩增出274、221、482、108 bp条带,无非特异性扩增,4种病原菌检出限达到10~100 CFU/mL;对17份人工染菌牛奶样品进行检测,检出结果与国标培养法完全一致。该研究结果为快速、高效、准确地检测出乳品中单核细胞增生李斯特氏菌、蜡样芽胞杆菌、鼠伤寒沙门氏菌、大肠埃希氏菌O157∶H7提供了一种实用方法,也为其他食源性致病菌的快速检测提供了思路。

应用综合干预措施对促进住院患者抗菌药物治疗前病原学送检的效果评价

目的探讨应用综合干预措施对促进住院患者抗菌药物治疗前病原学送检的效果。方法选取2020年1月—2021年12月某三甲医院治疗性使用抗菌药物的住院患者为研究对象,2021年1月开始实施综合干预措施,比较干预前组(2020年1—12月)和干预后组(2021年1—12月)抗菌药物治疗前病原学送检率、送检分类和重点监管科室送检率。结果共纳入治疗性使用抗菌药物的住院患者10239例,其中干预前组4526例,干预后组5713例。干预后的抗菌药物治疗前、限制级抗菌药物治疗前、特殊级抗菌药物治疗前病原学送检率分别为94.56%、94.72%、96.03%,高于干预前的83.74%、84.47%、84.95%,差异均有统计学意义(均P<0.05)。干预后的指向性病原体送检率为64.87%,高于干预前的28.04%,差异均有统计学意义(均P<0.05)。干预后的重症医学科、呼吸与危重症医学科、儿科、神经外科和普通外科治疗性使用抗菌药物病原学送检率分别为93.20%、91.17%、92.20%、94.12%、91.15%,高于干预前的85.00%、82.19%、83.20%、83.33%、83.03%,差异均有统计学意义(均P<0.05)。结论应用综合干预措施可提高住院患者抗菌药物治疗前病原学送检率,应密切关注医院感染诊断相关病原学送检指标和无菌体液送检指标。

石墨烯新材料在畜禽生产中的应用研究进展

石墨烯被称为“二十一世纪新材料之王”,在化工、材料、医药等领域都表现出了广阔的应用前景。近年来,石墨烯新材料在畜禽养殖生产中也得到了广泛应用。笔者总结了石墨烯及其衍生物在畜禽繁殖、畜禽环境监测、畜禽产品有害物残留监测、病原体检测、畜禽免疫中的研究和应用情况,分析了石墨烯新材料的生物安全性,指出了石墨烯在畜禽生产领域应用中的问题,并且对石墨烯在畜禽生产领域的应用前景进行了展望,旨在为石墨烯在畜禽生产领域中的应用提供理论基础。

基于CRISPR/Cas系统的检测技术在老年感染性疾病常见病原体中的研究进展

老年感染性疾病是全球老年人死亡的主要原因之一。由于老年人免疫力下降,合并慢性基础疾病等因素,导致其感染因素更复杂且临床表现各异。及早、快速和准确地鉴别出老年感染性病原体,对临床精准的抗感染治疗,减少老年病人并发症及降低死亡率有重要意义。传统病原体检测方法存在依赖标本活菌,检测时间长,检测人员准入条件高或依赖昂贵仪器等缺点。随着CRISPR/Cas系统在微生物检测中的应用,基于CRISPR/Cas系统的检测技术在老年感染性疾病病原体应用的优势也日渐突出。CRISPR/Cas系统能高效准确地鉴定出病原体,尤其是对于标本量少、微生物丰度低、采样困难或需要快速床旁检测的老年感染性疾病的辅助诊断具有重要的价值。本文主要对基于CRISPR/Cas系统的检测技术在老年感染性疾病病原体检测方面的研究进展进行综述。

病原体宏基因检测在器官捐献患者中的应用

目的探讨病原体宏基因组二代测序(metagenomic next-generation sequencing,mNGS)应用于器官捐献者病原微生物的检测,为器官移植后的抗感染药物靶向治疗提供依据。方法回顾性分析了武汉大学中南医院于2021年8月至2023年6月142例器官捐献者的临床病例,收集捐献者的血液样本的mNGS检测和病原微生物培养检测结果,对其进行分析比较。结果142例捐献者中98例在mNGS中检测到病原微生物,阳性检出率为69.01%。其中,59例(60.20%)为单纯病毒感染,12例(12.24%)为单纯细菌感染,4例(4.08%)为单纯真菌感染,混合感染23例(23.47%)。共检测出病原微生物173株。其中,病毒125株(72.25%),细菌40株(23.12%),真菌8株(4.62%)。对捐献者血液样本送检mNGS同时送检微生物培养检测,142例捐献者中20例(14.08%)血培养检测到病原微生物,共检测出21株病原微生物,其中20例(95.24%)为细菌,1例(4.76%)为真菌。器官保存液中培养出病原微生物2例(1.40%)。结论mNGS对病原微生物的检出率高于微生物血培养检测。mNGS检测和血培养病原微生物检测在细菌的诊断上差异不明显,而在检测真菌及病毒阳性时有统计学意义,且mNGS检测比血培养病原微生物检测能大幅度缩短检验时长。

分子生物学技术在食品病原微生物检测中的应用探索

随着食品安全问题日益凸显,快速准确地检测病原微生物显得尤为重要。分子生物学技术以其灵敏度高、特异性强、检测时间短等优势,在食品病原微生物检测中展现出巨大的应用潜力。本文首先介绍分子生物学技术在食品病原微生物检测中的应用意义,然后分析应用存在的问题,最后提出有效的应对策略。未来,随着技术的不断进步与创新,分子生物学技术在食品安全检测领域将发挥更加重要的作用。

常见食源性病原菌的快速检测技术研究进展

食源性病原菌广泛存在于自然环境中,可通过肉类、海产品、奶制品等多种媒介传播,引起食源性疾病的发生。目前检测食源性病原菌的方法主要以平板培养辅以生化鉴定和血清型鉴定为主,检测的周期较长,效率较低,因此建立快速、精准的食源性病原菌检测方法对常见病原菌的现场检测及疾病的快速诊断尤为重要。本文对近年来常见食源性病原菌的快速检测方法进行了概述,主要包括免疫学检测手段、分子生物学检测方法和生物传感器检测技术,比较其优缺点,着重介绍了可以实现现场检测与实时检测的新兴技术:核酸-微流控检测技术,以期为常见食源性病原菌的预防与检测提供相关理论参考。

重组酶介导等温扩增技术(RAA)在病原微生物检测中的应用进展

重组酶介导等温扩增(RAA)是一种恒温快速核酸扩增技术,是重组酶等温扩增技术中具有代表性和前沿性的检测技术,其弥补了现有核酸体外扩增技术需要热循环的局限性,使得核酸体外扩增更加简便快捷,目前已被广泛应用于动物疫病检测。本文介绍了RAA技术的检测原理、引物探针设计及优缺点,综述了其在病毒、细菌、寄生虫及抗生素耐药基因检测等方面的应用进展,总结了国内现有RAA技术标准及检测产品开发情况,以期为拓展RAA技术的应用提供参考。

白带常规与阴道炎六联检在阴道感染性疾病病原体检测中的价值

目的探讨白带常规及阴道炎六联检对阴道感染性疾病中病原体的检测价值。方法以2022年11月—2023年4月在厦门市第三医院接受妇科检查的500例白带异常患者为观察对象,均先后接受白带常规和阴道炎六联检。对比分析单一方法检查和联合检查在阴道感染病原体检测和临床诊断中的效能。结果白带常规检查显示,白细胞(white blood cell,WBC)计数增高141例(28.2%),细菌性感染91例(18.2%),念珠菌感染12例(2.4%),滴虫性感染15例(3.0%),阴道炎检出259例(51.8%);阴道炎六联检显示,WBC计数增高156例(31.2%),细菌性感染95例(19.0%),念珠菌感染35例(7.0%),滴虫性感染28例(5.6%),阴道炎检出314例(62.8%);2项联合检查显示,WBC计数增高158例(31.6%),细菌性感染117例(23.4%),念珠菌感染43例(8.6%),滴虫性感染40例(8.0%),阴道炎检出358例(71.6%),联合检查的细菌性感染、念珠菌感染、滴虫性感染的检出率及阴道炎检出率高于单一检查(P<0.05)。白带常规、阴道炎六联检及联合检测的白带清洁度比较,差异有统计学意义(P<0.05)。结论白带常规联合阴道炎六联检可提高阴道感染性疾病病原体检出率。

鸡群中鸡传染性贫血病原与抗体检测及分离株VP1基因序列分析

为了解鸡群中鸡传染性贫血病毒(CAV)感染及抗体产生情况,从黑龙江省两个无临床症状鸡群(B群和S群)中,分别采集血清通过ELISA方法进行抗体检测,采集抗凝血并分离淋巴细胞,针对基因组保守序列设计引物,通过PCR方法进行病原检测,并对从B群病原阳性鸡分离到的分离株进行VP1基因序列分析。结果显示,B群和S群CAV抗体阳性率分别为62.7%(79/126)和68.2%(88/129),病原阳性率分别为43.0%(49/114)和62.3%(48/77),其中,B群和S群中抗体和病原双阴性的鸡分别为23.7%(27/114)和15.6%(12/77),而抗体和病原双阳性的鸡分别高达28.1%(32/114)和46.8%(36/77);在双阳性鸡中,B群和S群分别有62.5%(20/32)和58.3%(21/36)抗体滴度在1.0×10^(4)及以上,从B群病原阳性鸡全血中分离到一株CAV(HLJ/2022株),分离株HLJ/2022第394位氨基酸为谷氨酰胺,具有强毒的分子特征;两个鸡群的环境拭子CAV阳性率为10.0%(3/30),存在于消毒前的鸡蛋表面、更衣处和鞋底。研究表明,检测鸡群的CAV抗体阳性率在62.7%以上,病原阳性率在43.0%以上,抗体滴度在1.0×104及以上CAV仍可存在,CAV流行株(HLJ/2022)具有强毒分子特征,鸡群环境中存在CAV污染,结果为CIA防控提供重要的参考意见。