One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Study on Distribution of Four Pseudomonas Species in Living Environment Using Multiplex PCR

Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.

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。

香蕉枯萎和细菌性软腐病菌的多重PCR检测

香蕉枯萎病菌Fusarium oxysporum f.sp.cubense和细菌性软腐病菌Dickeya zeae的复合侵染为害给香蕉产业发展带来严重挑战,有必要建立相关病害的多重聚合酶链式反应(multiplex polymerase chain reaction, multiplex PCR)检测技术。本文基于尖孢镰刀菌古巴专化型1号生理小种(F.oxysporum f.sp.cubense race 1,FOC1)基因组contig 438区间(35 631-37 693 bp)(GenBank:AMGP01000438.1)和4号生理小种(F.oxysporum f.sp.cubense race 4,FOC4)基因组contig 195区间(4 028-6 126 bp)(GenBank:AMGQ01000195.1)存在160 bp插入序列差异设计特异扩增引物FOC-F/-R,同时以香蕉细菌性软腐病菌D.zeae的促旋酶B亚单位基因(the subunit B of gyrase gene)(GenBank:JQ284039)序列设计特异扩增引物gyrB-F/-R。多重PCR检测结果显示:本技术可在一次PCR扩增反应内同时检测香蕉枯萎病菌1号、4号生理小种和细菌性软腐病菌;多重PCR的灵敏度结果表明:检测香蕉枯萎病菌的DNA浓度最低限为0.1 ng/μL,细菌性软腐病菌的灵敏度为103cfu/mL;检测结果稳定可靠。因此,本研究建立的多重PCR检测方法可有效应用于检测香蕉发病组织中的香蕉枯萎病菌和细菌性软腐病菌,也可用于香蕉种苗和田间土壤带病菌的监测,为香蕉种植保驾护航。

TaqMan多重实时定量PCR快速检测3种常见食源性病原菌

建立一种可同时快速检测大肠杆菌O157:H7、单增李斯特菌和蜡样芽孢杆菌的多重实时定量PCR(qPCR)方法。依据大肠杆菌O157:H7 tir基因、单增李斯特菌mpl基因和蜡样芽孢杆菌entFM基因的保守序列分别设计特异性引物和TaqMan探针,建立多重qPCR反应体系,进行灵敏度、特异性和稳定性试验,同步检测人工染菌牛奶样品中的病原菌并与国家标准方法作对比。结果表明,建立的多重qPCR方法灵敏度高,最低检出限为12 CFU/mL;特异性强,只对3种目标菌进行PCR扩增;稳定性好,各重复性试验中Ct值的变异系数<1%;所有受污染样品阳性检出率均为100%,与国家标准方法检测结果一致,且检测周期缩短至6 h。本研究建立的TaqMan多重qPCR方法能同时快速、准确地检测乳品中的大肠杆菌O157:H7、单增李斯特菌和蜡样芽孢杆菌,为食品安全提供技术支撑。

4种常见食源性病原菌多重PCR检测技术研究

为了解决常规方法检测食源性病原菌存在操作繁琐、周期长的问题,基于4种常见食源性病原菌特异基因序列扩增提出了一种快速、高效的多重PCR(multiplex polymerase chain reaction, MPCR)检测方法。根据单增李斯特菌iap基因、蜡样芽孢杆菌gyrB基因、产志贺毒素大肠埃希氏菌stxⅠ基因、肠炎沙门氏菌invA基因的特异序列分别设计引物后建立MPCR反应体系,测试其特异性、敏感性和可行性,并与国标培养法进行比较。结果表明:MPCR扩增出4个目标基因的特异性条带大小依次为371、221、432、171 bp;在58℃的退火温度下,MPCR扩增表现出良好的特异性,无非特异性扩增,4种病原菌最低检出限达到102CFU/mL;MPCR方法和国标培养法对多种食源性病原菌感染的牛奶样品的检测结果完全一致,检测周期由原来的5~7 d缩减到8~9 h。所建立的MPCR技术作为一种快速、高效的检测方法,可为食品安全生产提供保障。

基于新型高保真Taq酶的多重等位基因特异性PCR方法的建立与应用

在8号染色体存在部分替换片段的小鼠上挑选3个SNP位点进行初步方案检验,包括高保真Taq酶的特异性测试以及引物浓度对分型结果的影响,并评价该方法的灵敏度;之后应用该技术检测野生1号染色体替换系小鼠上的9个SNP的不同样本。对Taq酶的特异性研究表明:引物在未引入突变的前提下检测结果与测序结果保持一致;引物稀释实验说明浓度最低为0.003~0.006μmol/L,灵敏度的检测结果表示最低浓度检测限可达0.07 ng/μL以下。最后检测得到5种标准样本的基因型,其代表9个SNP的组合。成功验证并建立该新型高特异性Taq酶应用于多重等位基因特异性PCR实验的总体流程。提供一套具备高特异性、能够针对多个SNP位点进行检测的低成本方案,对多重等位基因特异性PCR技术的开发同样具有参考价值。

H1-SIV、H3-SIV、NA-PRRSV和EU-PRRSV多重PCR检测方法的建立及应用

猪繁殖与呼吸综合征病毒(PRRSV)感染肺泡巨噬细胞,严重损害免疫系统,导致与其他病毒共感染现象,其中猪流感病毒(SIV)也是病原之一。为了快速检测和区分PRRSV基因型与SIV亚型,本研究针对H1、H3亚型SIV的HA基因和美洲型、欧洲型PRRSV的N、M基因的保守序列,分别设计了4对特异性扩增引物,在引物浓度、退火温度等反应条件的系统优化下,分别对其特异性和敏感性进行验证,建立了能够快速检测以上4种病原的多重PCR方法,并对155份临床样品进行了检测。结果显示,该检测方法特异性好、灵敏度高,对H1-SIV、H3-SIV、NA-PRRSV和EU-PRRSV的最低检出量均低至9.86×10^(0) copies/μL。以单一PCR方法对所有临床样品进行检测后发现,检测结果与多重PCR检测结果一致,证明这种多重PCR方法可以用于这4种病毒的快速检测与分型鉴定。

野生型非洲猪瘟病毒多重数字PCR方法的建立

为建立一种鉴别诊断野生株和基因缺失株非洲猪瘟病毒(African swine fever virus,ASFV)的方法,针对ASFV的B646L、EP402R、DP96R基因保守序列设计3套引物和探针,优化三重荧光定量PCR体系作为基础建立微滴式数字PCR(droplet digital PCR,dd PCR)检测方法,并对方法的特异性、灵敏性及重复性进行评估。结果显示,优化后的多重荧光定量PCR检测方法的标准曲线线性关系良好,对PRRSV、PEDV、PRV、VSV、PCV、RNase-free水、重组质粒标准品进行特异性检测,特异性良好;重复性试验结果显示,变异系数均小于2%;对3种重组质粒的最低检测下限均为102 copies/μL。参照上述多重荧光定量PCR方法优化后的条件,进行多重ddPCR检测方法的建立,并且对该方法进行评价。结果显示,ddPCR检测方法标准曲线的线性关系良好;特异性及重复性良好;最低检测下限B646L为11.6 copies/μL、EP402R为13.3 copies/μL、DP96R为16.9 copies/μL;ddPCR的检测灵敏度比荧光定量PCR有优势。使用建立的多重ddPCR方法对14份P3实验室中的样品进行实验室样品检测,样品检测符合率达到100%。成功建立了能够同时检测用于鉴别野毒株感染与疫苗株的荧光定量PCR和微滴式ddPCR方法。

柑橘3种病毒类病原多重RT-PCR检测技术的建立及应用

【目的】建立柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)、柑橘衰退病毒(citrus tristeza virus,CTV)和啤酒花矮化类病毒(hop stunt viroid,HSVd)的多重RT-PCR检测体系。【方法】设计多重RT-PCR引物,分析其特异性,确定其最佳浓度比、最适退火温度及灵敏度,在此基础上对福建地区的柑橘样品进行检测。【结果】确定了CYVCV-F/R、CTV-F/R和HSVd-F/R等3对引物的最佳浓度比例为1∶1∶2,最适退火温度为52.9℃,灵敏度结果显示该体系可检测模板稀释到10^(-2)的阳性样品。应用该体系对采自福建部分地区的157份柑橘样品进行检测,结果发现,CYVCV、CTV和HSVd的检出率分别为47.1%、56.7%和22.9%。【结论】成功建立了柑橘CYVCV、CTV和HSVd病原的多重RT-PCR检测方法,为该类病害的检测提供准确、快速的检测方法。

Study on Distribution of Acinetobacter baumannii Complex in Dental Hospital Using Multiplex PCR

Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex species is considered of critical importance to public health organizations. The reliable identification method able to distinguish A. baumannii from genetically close Acinetobacter species is needed, because these species are unable to be differentiated by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in dental hospital using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene and DNA gyrase subunit B (gyrB) of each species of A. baumanii complex species. Swab samples were collected from ten dental spittoon units in dental hospital, and the distribution of A. baumanii complex species was investigated using a multiplex PCR. Results: These primers were able to distinguish each species of A. baumanii complex species clearly. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Multiplex PCR for Identification and Detection of Cassava Mosaic Begomoviruses in Togo

Cassava mosaic disease (CMD) caused by Cassava Mosaic Begomoviruses (CMBs) is one of the most devastating crop diseases and a major constraint for cassava production. In order to ensure surveillance for epidemic prevention, low-cost diagnostic tools are appropriate for large-scale testing of cassava viruses. Multiplex PCR diagnosis is one approach that can reduce diagnostic costs and delays. A multiplex PCR approach was developed for simultaneous detection of African cassava mosaic virus (ACMV), East African Cassava Mosaic Virus and East African cassava mosaic Cameroon virus (EACMV/CM) in Togo CMD-infected cassava leaves. Three primers pairs were used to target their respective viruses in a single tube PCR. Multiplex PCR detected ACMV, EACMV and EACMV/CM in plant DNA extracts prepared from cassava leaves infected with CMB. The primers amplified 783 bp specific to ACMV, 650 bp specific to EACMV and 560 bp specific to EACMCV/CM in both uniplex and multiplex formats. Multiplex PCR is an excellent tool for the effective control of cassava diseases. .

One Step Multiplex PCR for Identification of Candida haemulonii complex and Candia auris

Purpose: Recently, Candida haemulonii complex (Candida haemulonii, Candida duobushaemulonii and Candida haemulonii var. vulnera) and two genetically close species (Candida pseudohaemulonii and Candida auris) have emerged as an opportunistic fungal pathogen associated with various infectious diseases of humans, and most of those isolates have displayed antifungal resistance. Because it is difficult to differentiate these microorganisms by a current technique, unfortunately, it is important to establish a method for identifying them accurately. The purpose of the present study was to design species-specific primers in order to identify and detect C. auris, C. pseudohaemulonii, and C. haemulonii complex, i.e. , C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 26S rRNA, 18S rRNA, and RPB1 genes and ITS region of five Candida species. Results: The multiplex PCR method developed in this study was able to distinguish five Candida species clearly. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction.

福建省茶树病毒种类鉴定及多重PCR检测技术的建立

【目的】明确福建省茶树主要病毒种类和分布情况,并建立同时快速检测多种病毒的多重PCR检测技术。【方法】2019—2023年,从福建省福州、南平、宁德、泉州、漳州、厦门、三明、莆田和龙岩9个地市区采集具有褪绿、皱缩和坏死等疑似病毒感染症状的茶树样品1869份,采用高通量测序技术结合PCR和RT-PCR检测的方法对茶树病毒病的病原进行鉴定,并将PCR扩增获得的特异性目的片段进行克隆和测序,对获得的序列进行系统发育分析;同时,根据GenBank上已报道的病毒序列设计特异性引物,通过退火温度、引物浓度、循环数等反应条件和反应程序的优化,建立同时检测福建茶树主要病毒的多重PCR检测技术,并测定该技术的特异性、灵敏度及实际应用效果。【结果】从所采集的茶树病样上检出3种病毒,按检出率从高到低依次为油茶双生病毒(oil tea associated geminivirus,OTaGV)(48.90%)、茶树坏死环斑病毒(tea plant necrotic ring blotch virus,TPNRBV)(26.75%)和茶树潜隐病毒1(camellia cryptic virus 1,CCV1)(17.98%);在检出病毒的1258份样品中,有807份为OTaGV、TPNRBV或CCV1单独侵染,检出率分别为37.20%、21.38%和5.56%;其余451份样品为2种或3种病毒复合侵染,复合侵染检出率达35.85%,OTaGV+CCV1、TPNRBV+CCV1、TPNRBV+OTaGV、OTaGV+CCV1+TPNRBV 4种类型复合侵染检出率分别为17.49%、0.40%、14.71%、3.26%。在地理分布上,OTaGV在福州、南平、宁德、泉州、漳州、厦门、三明、莆田和龙岩9个地区均有分布,其中漳州OTaGV检出率最高,为96.77%;CCV1在福州、南平、宁德、泉州、漳州、三明、莆田和龙岩8个地区均有分布,其中三明CCV1检出率最高,为66.00%;TPNRBV在福州、南平、宁德、泉州和漳州5个地区均有分布,其中泉州TPNRBV检出率最高,为79.13%;在福建省9个地区中,厦门地区仅检出OTaGV,三明、莆田和龙岩地区检出OTaGV和CCV1,其他5个地区同时检出OTaGV、TPNRBV和CCV1;福州、南平、宁德、泉州、漳州、三明、莆田7个地区检测到病毒复合侵染,其中漳州地区病毒复合侵染检出率最高(85.00%)、宁德地区病毒复合侵染检出率最低(23.03%)。利用测定的CCV1和TPNRBV部分基因序列构建系统发育树,结果表明本研究获得的CCV1分离物(FW)与已报道的福建分离物FJ-SH104(GenBank登录号:ON807095)亲缘关系最近、TPNRBV分离物(FU)与福建分离物QZHA92(GenBank登录号:OQ948454)亲缘关系最近。经优化建立的多重PCR检测技术特异性强,仅OTaGV、TPNRBV和CCV1能扩增出特异性目的片段,而其他病毒及健康茶树样品上均未扩增出特异性目的片段;多重PCR灵敏度最低可以检测到稀释至10^(-4)倍的OTaGV、TPNRBV和10^(-3)倍的CCV1;利用该多重PCR检测技术对茶园中采集的60份病样进行检测,检测结果与单一PCR检测结果完全相符。【结论】OTaGV、TPNRBV和CCV1是当前福建茶树上主要病毒种类,其中OTaGV为福建地区首次报道,该病毒可侵染茶树也为首次发现;在检出的3种病毒中,以OTaGV发生分布范围最广,其次为CCV1、TPNRBV;福建地区茶树病毒目前仍以单独侵染为主,但同时存在较多的复合侵染,复合侵染类型包括TPNRBV+CCV1、TPNRBV+OTaGV、CCV1+OTaGV和OTaGV+CCV1+TPNRBV;建立的多重PCR检测技术特异性强、灵敏度高,可用于茶园茶树上OTaGV、CCV1和TPNRBV3种病毒的快速检测。研究结果可为福建茶树病毒病的综合防控提供理论依据和技术支持。

病原体多重PCR检测技术研究进展

引起某一症候群传染病病原体种类繁多,且可能存在复合感染,快速、准确地诊断病原体,既可有效预防疾病传播,也可以很好地指导临床用药。多重PCR(multiplex polymerase chain reaction,MPCR)技术,可在一个反应体系中同时检测多个病原体,满足突发公共卫生事件中迅速查明病因需要。根据MPCR产物检测信号的时机不同,MPCR技术可分为PCR反应实时检测、终点检测、反应后检测3种类型。本文主要针对这3种类型的MPCR技术研究进展进行综述,并探讨此类技术的发展方向。

Rapid Detection Co-infections of Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus by One-step Multiplex RT-PCR

Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.

Development and Validation of Multiplex One-Step Real-Time TaqManqRT-PCR Assays for Detection and Quantification of Arboviral Encephalitis Viruses

Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.

蜂蜜中转基因成分启动子和终止子的多重实时荧光PCR法检测

为能高效率定位蜂蜜中的转基因可疑阳性样品,以10种转基因启动子和终止子(花椰菜花叶病毒35S启动子(pCaMV35S)、农杆菌的胭脂碱合成酶基因终止子(tNOS)、玄参花叶病毒的35S启动子(p FMV35S)、农杆菌的胭脂碱合成酶基因启动子(pNOS)、玉米泛素基因的启动子(pUbi)、烟草的花蕊特异性TA29的基因发育调节启动子(pTA29)、豌豆二磷酸核酮糖羧化酶基因的终止子(tE9)、章鱼碱合成酶终止子(t OCS)、谷氨酰胺转移酶7终止子(tg7)、花椰菜花叶病毒终止子(tCaMV35S))为研究靶标,进行10种启动子和终止子的引物和探针组合筛选、反应条件优化、灵敏度测定、质控品测试比对等实验,建立3组多重实时荧光PCR反应体系,并将其应用于市售蜂蜜的检测。结果表明:建立的3组多重实时荧光PCR反应体系能精准检出目的基因,其灵敏度分别为0.01、0.04、0.04 ng/μL;14种转基因质控品的测试显示多重实时荧光PCR和单重实时荧光PCR的检测结果一致;40份市售蜂蜜的筛查中,有两份蜂蜜检出pCaMV35S和tNOS,其余未检出,该方法大大提高了检测效率。该方法为蜂蜜中转基因成分的检测提供了快筛快检技术,同时也适用其它产品转基因成分的检测。

卵形巴贝斯虫与中华泰勒虫双重PCR检测方法的建立

为建立一种能快速对卵形巴贝斯虫(Babesia ovata)和中华泰勒虫(Theileria sinensis)同时进行检测的双重PCR方法。根据GenBank已报道的卵形巴贝斯虫AMA1基因和中华泰勒虫MPSP基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。结果显示:双重PCR可特异扩增出卵形巴贝斯虫和中华泰勒虫目的条带,片段大小分别为500 bp和986 bp。该方法具有较好的特异性,对卵形巴贝斯虫和中华泰勒虫的最低检出浓度为16 fg/μL。对采集的90份牛血液样本进行双重PCR检测,卵形巴贝斯虫阳性率为30%(27/90),中华泰勒虫阳性率为16.67%(15/90),混合感染率为10%(9/90)。结果表明,双重PCR方法可用于卵形巴贝斯虫和中华泰勒虫的快速诊断和流行病学调查。

猪呼吸道病原菌多重PCR检测方法的建立及应用

为建立一种快速诊断由猪链球菌(SS)、猪胸膜肺炎放线杆菌(App)、猪多杀性巴氏杆菌(Pm)、副猪嗜血杆菌(Hps)、猪支气管败血波氏杆菌(Bb)和猪肺炎支原体(Mhp)单独或混合感染引起的猪呼吸道疾病的检测方法,基于上述6种病原菌对应的gdh(SS)、apxⅣA(App)、KMT1(Pm)、16S rRNA(Hps)、fla(Bb)和p36 ldh(Mhp)基因设计特异性引物,通过优化各反应条件建立了能同时检测SS、App、Pm、Hps、Bb和Mhp的多重PCR方法。结果显示,该方法特异性强,仅对SS、App、Pm、Hps、Bb和Mhp有特异性扩增,对大肠埃希氏菌、奇异变形杆菌、停乳链球菌和金黄色葡萄球菌等其他病原菌的扩增结果均为阴性;敏感性较高,对6种病原菌DNA的最低检出限分别为SS 8.66×10^(2)拷贝/μL、App 1.05×10^(4)拷贝/μL、Pm 8.61×10^(2)拷贝/μL、Hps 9.01×10^(2)拷贝/μL、Bb 7.54×10^(2)拷贝/μL、Mhp 3.86×10^(3)拷贝/μL。利用该多重PCR和各单项PCR分别检测贵州省9个市(州)部分猪场的469份临床样品,结果显示,多重PCR阳性检出率为App 0.85%(4/469)、Pm 15.35%(72/469)、SS 59.49%(279/469)、Hps 52.67%(247/469)、Mhp 6.40%(30/469)、Bb 5.12%(24/469)。混合感染主要以SS和Hps为主,阳性检出率为30.92%(145/469),其次是Pm、SS和Hps混合感染,阳性检出率为7.04%(33/469),其他混合感染情况占比较低。多重PCR和单项PCR的总阳性符合率为96.76%。结果表明,建立的多重PCR检测方法为SS、App、Pm、Hps、Bb和Mhp混合感染的临床快速检测及流行病学调查提供了技术支持。