Genotyping of Toxoplasma gondii in Sheep and Cattle Meat Using PCR-RFLP Technique

distribution.The main source of infection for humans is livestock and meat-producer animals.The relationships between Toxoplasma genotype and biological characteristics of the parasite have already been identified.According to the pathogenicity of the parasite in laboratory animals,Toxoplasma is divided into three genotypes included type I,II and III.Understanding the genotype of the parasite,could help us to predict clinical features and severity of disease.The aim of this study was to identify genotypes of T.gondii in cattle and sheep meat and meat products in Ahvaz city southwest of Iran.One hundred and ninety samples of tongue,heart and muscles of sheep and cattle and meat products,including sausages and burgers,were collected from slaughterhouses and stores.To identify Toxoplasma gondii,DNA were extracted from samples and B1 gene were amplified by specific primers.To determine the genotype of T.gondii,PCR-RFLP was done on positive samples using by amplifying GRA6 gene and endonuclease Msel enzyme.Data analysis showed that the strain of the parasite in all positive samples belonged to genotype I.In this study the predominant Toxoplasma genotype was type I which can cause severe clinical symptoms in immunocompromised patients.Further research is needed to determine the genotype of the parasite in humans and other animals.

Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus

AIM: To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS: Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS: Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 (33%), 41 (33%) and 45 (36%) samples, and genotype C in 76 (60%), 76 (60%) and 81 (64%) samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real-time PCR was the rapidest and cheapest among the three assays. CONCLUSION: Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.

Two-Tube Multiplex PCR for Genotyping Tuberculous and Nontuberculous Mycobacterial Species in Pathology Specimens

Background: The incidence of mycobacterial infection, in particular M. tuberculosis complex (MTC), is increasing in some Western countries, while nontuberculous mycobacteria (NTM) may be recognized more frequently in clinical specimens worldwide. The clinical scenario and available histopathology alone are often insufficient to separate these two categories of mycobacterial disease, whose behavior and treatment differ. In particular, NTM may be clinically unsuspected in pathological specimens and the opportunity for culturing missed. Methods: We developed two multiplex PCR assays, which distinguish MTC from NTM by detecting the IS6110 insert in the first tube and discriminating up to 14 NTM reference strains in the second by targeting the 16S-23S rRNA internal transcribed spacer. Test material included 594 routine clinical specimens with diverse pathology;many were granulomas unrelated to mycobacterial infection. About 75% were formalin-fixed paraffin blocks, the remainder mainly cytologic imprints or aspirates on FTA cards submitted on suspicion of mycobacterial infection either to avoid frozen sectioning (with the attendant risk of aerosolisation) or at the time of fine needle aspiration. Results: The paraffinized material yielded 53 MTC positives and the cytological 21 positives. A subset consisting of 337 specimens was also analyzed for NTM and yielded 51 positives. The frequency of simultaneous NTM infection in tuberculous patients was about 17%. Mycobacterium avium complex represented the dominant NTM species overall, showed a predilection for lung and lymph node, and together with M. haemophilum were the second most frequent NTM just behind M. ulcerans/M. marinum in skin and soft tissue, the category displaying the largest NTM diversity. Conclusions: Cytological and deparaffinized tissue analyzed in a new two-tube multiplex PCR allows for specific discrimination of causative agents in mycobacterial infection. MTC is readily distinguished from NTM for appropriate therapy, and NTM presumptively diagnosed at the species level allows appropriate choices of antimicrobials.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

十八微卫星教材对以前为 Populus tremuloides Michx 在橡树理兹公民实验室发展了。并且 Populus trichocarpa Torr。与格雷在幼发拉底河白杨为扩大被屏蔽， Populus euphratica Oliv。13 loci 被发现表示从二到 17 等位基因的多型性。八很可变的 loci 被选择建立并且优化二复合聚合酶链反应(PCR ) 试金。总共包含 436 棵树的三张人口被用来描绘选择 loci 并且为出身分析查明他们的适用性， genotyping 学习。通过对 genotyped 树的性的同种细胞的身份的 cross-checking，我们为合并遗传型是不到 0.045 估计了最大的错误率。

基于Real-time PCR法检测乳粉中牛源性成分定量研究

基于Real-timePCR建立了乳粉中牛源性成分相对定量检测方法,并对牛的特异性引物与探针进行了特异性、灵敏度和稳定性测试。通过模拟不同浓度牛乳粉与马乳粉混合样本,根据其△Ct值的函数关系进行线性拟合进而绘制标准曲线,建立乳粉中牛源性成分的相对定量检测。结果显示,该方法的最低检测限为0.00001 mg/mL,回收率为91.11%~119.2%,组间变异系数≤0.58%、组内变异系数≤1.44%。说明该方法在特异性与稳定性上适用于乳粉中牛源性成分及含量的掺假检测。

一种基于real-time PCR技术的TTV检测方法的建立及应用

目的:本研究旨在开发一种具有更高灵敏度和特异性的TTV检测技术,为揭示TTV在多种疾病过程中的作用提供重要的技术支持。方法:为了更精确、灵敏的检测TTV,本研究分析了目前公布的所有亚型的TTV基因序列,在此基础上建立了一种基于UTR区域的real-time PCR检测方法,并与文献报道应用较为广泛的PCR检测方法进行了对比。结果:本研究建立的方法在1×10^(7)~1×10^(1) copies/μL标准品浓度范围内具有良好的线性关系,相关系数为1.000,斜率为-3.446,检测下限为1×10^(1) copies/μL。重复性试验结果显示,组内变异系数为7.22%,表明本方法重复性、稳定性较强。针对30份临床样本,使用本研究建立的real-time PCR检测方法及目前被多个研究所使用的4套引物进行对比。结果表明,本研究所建立的方法灵敏度显著高于文献中报道的4种方法(P<0.01);Sanger测序结果表明,本方法检测出的30份阳性样本均为TTV,检测特异性为100%。结论:本研究采用基于TaqMan探针的real-time PCR检测方法,检测灵敏性高、覆盖基因型范围广,尤其对于TTV病毒载量较低的情况下能够进行定量检测,对于TTV病毒的致病性及作为免疫标志物的应用提供重要的技术支持。

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.

24重荧光real-time PCR技术在食物中毒快速检测中的应用

目的:应用24重荧光real-time PCR检测技术快速筛检食物中毒病原菌,结合国家标准中的培养法探讨24重荧光real-time PCR检测技术符合性和应用价值。方法:采用高灵敏度、高特异性的24重荧光real-time PCR检测技术作为中毒病原菌的初筛方法,国标方法进行细菌分离培养,并对分离出的病原菌进行生化鉴定。结果:5份食物中毒患者肛拭子在增菌前检出4份霍乱弧菌核酸(非O1/非O139群,24重荧光real-time PCR),9份患者肛拭子在增菌后检出5株霍乱弧菌(非O1/非O139群,国标培养法),其中包含4份PCR技术初筛阳性样品,两个方法的符合率为80%。所有样本均未检出沙门氏菌、志贺菌、副溶血性弧菌、致泻性大肠埃希菌以及金黄色葡萄球菌。结论:应用24重荧光real-time PCR检测技术同时检测24种常见致病病原菌,能高效锁定中毒病原菌。将其与国标培养法相结合,对临床治疗和食物中毒快速处置能起到积极作用,值得应用和推广。

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

基于病菌孢子捕捉和real-time PCR技术的田间空气中小麦白粉病菌孢子动态监测及病情估计模型研究

利用Burkard定容式孢子捕捉器结合real-time PCR定量技术,分别对种植高抗、中感和高感白粉病小麦品种的田间空气中白粉病菌分生孢子浓度进行监测,结果表明,real-time PCR定量与传统的显微观察计数两种方法测得的孢子浓度呈显著正相关(P≤0.01),且两种病菌孢子计数方法在同一抗性品种上监测到的孢子浓度动态相近。此外,两种方法测得的孢子浓度与各气象因子的相关性分析结果一致,空气中的白粉病菌孢子浓度主要与空气相对湿度显著正相关。在此基础上,利用两种方法测定的田间空气中白粉病菌孢子浓度分别建立了基于累积孢子浓度的田间病情估计模型。分析发现,基于两种孢子浓度测定方法建立的病情估计模型间无显著性差异,表明real-time PCR定量技术测定的孢子浓度在构建白粉病病情估计模型上具有一定可行性。该结果为real-time PCR定量技术与病菌孢子捕捉技术相结合用于小麦白粉病的监测和预测提供理论依据。

葡萄浆果内坏死病毒RT-q PCR检测技术建立及其在葡萄砧木中的时空分布规律

【背景】葡萄浆果内坏死病毒(grapevine berry inner necrosis virus,GINV)是近年来在中国报道的一种正单链RNA病毒,该病毒发生普遍且危害严重。高灵敏的检测技术是病毒田间监测和无病毒苗木培育的关键。【目的】建立灵敏度较高的GINV逆转录实时荧光定量PCR(reverse transcription real-time quantitative PCR,RT-qPCR)检测体系;明确不同葡萄砧木对GINV的敏感性;明确GINV在寄主植株中的时空分布规律,为该病毒的监测预警提供技术支撑。【方法】根据GenBank已登录GINV的复制酶(replicase,RP)、移动蛋白(movement protein,MP)和外壳蛋白(coat protein,CP)基因保守序列设计6套引物,通过常规RT-PCR和RT-qPCR筛选特异性强、扩增效果好的引物。再通过对退火温度和引物浓度等反应条件的优化建立GINV的SYBR Green I染料法RT-qPCR检测体系,并进一步对该技术的灵敏度、特异性和田间适用性进行评价。将GINV接种到贝达、SO4、101-14、140R和1103P 5种葡萄砧木上进行症状观察和病毒检测,以筛选GINV敏感性较高的指示植物。基于所建立的RT-qPCR技术对接种GINV葡萄砧木的不同生长时期、不同部位样品进行GINV检测,从而明确GINV在不同葡萄砧木的时空分布规律。【结果】建立了GINV SYBR Green I染料法RT-qPCR检测技术体系,其最佳引物为GINVRPYGF2/R2,最佳引物浓度为300 nmol·L^(-1),最佳退火温度为58.4℃。该技术对GINV的检测特异性强,其检测灵敏度达常规RT-PCR的1 000倍。症状观察结果表明贝达感染GINV的症状最为严重,表现为叶片系统性坏死,而其他砧木叶片仅表现褪绿斑驳和环斑症状。RT-qPCR检测结果表明GINV在EL27时期(坐果期)的相对含量最高,5个品种间的病毒相对含量在EL12(花序分明期)和EL27时期间无显著差异,EL31时期(果实增大期)的贝达与SO4、101-14、140R和1103P的病毒相对含量存在显著差异;GINV的相对含量在不同组织部位存在较大差异,由高到低依次为下部叶片、上部叶片、上部茎秆、下部茎秆、根部。【结论】建立了灵敏度高、特异性强的GINV RT-qPCR检测方法,利用该方法明确了GINV在不同葡萄砧木的时空分布规律。

国产化Y-STR试剂盒与YFiler^(TM) Platinum PCR扩增试剂盒的法医DNA检验性能比较研究

该文评估4种国产化Y-STR试剂盒与Yfiler^(TM) Platinum PCR扩增试剂盒的法医DNA检验性能,并进行比较分析。结果显示,4种国产化Y-STR试剂盒均可基本满足日常案件检测需要,在灵敏度和拮抗抑制剂的适用性的方面,SureID■ Y40 PCR扩增试剂盒和炎黄Y数据分型盒表现较优,在降解样本的适用性方面,Yfiler^(TM) Platinum PCR扩增试剂盒和国产化炎黄Y数据分型盒表现最优。

肉中猪源性成分Real-time PCR定量检测技术

【目的】建立一种快速、准确的肉中猪源性成分定量检测方法。【方法】首先从GenBank数据库中筛选猪特异性的微卫星DNA,根据微卫星DNA核酸序列设计引物,对常见10种动物基因组DNA进行PCR扩增,通过有无扩增产物判断筛选的微卫星DNA对猪源性成分的特异性。然后根据微卫星DNA核酸序列,设计特异性引物和探针,建立猪源性成分Real-time PCR检测方法,采用双标准曲线分别对猪源性成分和总动物源性成分进行定量,计算猪源性成分的百分含量。【结果】筛选到猪特异性微卫星DNA(Accession EF172428),根据其序列设计的引物SEQ-sus2-F/R只能从猪基因组DNA中扩增出目的条带,其他动物的基因组均无目的条带扩增。建立的Real-time PCR检测方法灵敏度为0.02 ng/25μL反应体系。该方法能够准确检测出混合DNA样品中猪源性成分和混合肉样品中猪源性成分,百分误差分别约为1.32%和1.06%-7.12%。【结论】本研究利用Real-time PCR技术建立的定量猪源性成分的检测方法可以用来检测猪源性成分在混合样品中的百分含量。

美国引进甘蔗热带种的ARMS PCR真实性鉴定

甘蔗遗传基础狭窄已经极大地阻碍了其育种进程,挖掘优异种质资源、创新亲本材料将成为未来甘蔗育种的重要方向之一。利用四引物扩增受阻突变PCR体系(ARMS PCR)对国家甘蔗种质资源圃保存的美国引进的24份疑似热带种材料进行真实性鉴定。PCR鉴定结果表明,供试材料中有‘Green German’、‘Black Fiji’、‘Chittan’、‘IN 84-072’和‘NG 21-002’这5份材料扩增带型与热带种‘拔地拉’(Badila)一致,仅出现428 bp的共有条带和278 bp的热带种特有条带,属于热带种带型;而其它19份材料同时具有278 bp的热带种特有条带和203 bp的割手密特有条带,属于热带种与割手密的杂交种带型。经过上述分子鉴定得出结论,24份美国引进的甘蔗种质中有‘Green German’、‘Black Fiji’、‘Chittan’、‘IN 84-072’和‘NG 21-002’这5份材料为热带种材料,建议后续加强其农艺性状和抗性调查,并作为甘蔗热带种新血缘亲本进行杂交利用。

A prolific and robust whole-genome genotyping method using PCR amplification via primer-template mismatched annealing

Whole-genome genotyping methods are important for breeding.However,it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species.In our study,we accidently discovered that in adapter ligation-mediated PCR,the amplification by primertemplate mismatched annealing(PTMA)along the genome could generate thousands of stable PCR products.Based on this observation,we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing(FBI-seq)using one specific primer,in which foreground genotyping is performed by primer-template perfect annealing(PTPA),while background genotyping employs PTMA.Unlike DNA arrays,multiple PCR,or genome target enrichments,FBI-seq requires little preliminary work for primer design and synthesis,and it is easily adaptable to different foreground genes and species.FBI-seq therefore provides a prolific,robust,and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the postgenomics era.

iBP-seq:An efficient and low-cost multiplex targeted genotyping and epigenotyping system

Inter-and intra-specific variations in phenotype are common and can be associated with genomic mutations as well as epigenomic variation.Profiling both genomic and epigenomic variants is at the core of dissecting phenotypic variation.However,an efficient targeted genotyping and epigenotyping system is lacking.We describe a new multiplex targeted genotyping and epigenotyping system called improved bulked-PCR sequencing(iBP-seq).We employed iBP-seq for the detection of genotypes and methylation levels of dozens of target regions in mixed DNA samples.iBP-seq can be adapted for the construction of linkage maps,fine mapping of quantitative-trait loci,and detection of genome editing mutations at a cost as low as$0.016 per site per sample.We developed an automated bioinformatics pipeline,including primer design,a series of bioinformatic analyses for genotyping and epigenotyping,and visualization of results.iBP-seq and its bioinformatics pipeline,available at http://zeasystemsbio.hzau.edu.cn/tools/ibp/,can be adapted to a wide variety of species.

多主棒孢SdhB-H278Y突变位点real-time PCR检测体系的建立与应用

根据GenBank已登录序列中黄瓜多主棒孢琥珀酸脱氢酶B亚基(SdhB)基因序列差异,针对SdhB-H278Y突变设计特异性引物,建立SdhB-H278Y突变实时荧光定量PCR(real-time PCR)检测体系。结果表明:供试多主棒孢携带SdhBH278Y、SdhB-I280V突变;SdhB-H278Y突变株对啶酰菌胺抗性较强,EC50值为21.47μg·mL^(-1)或>30μg·mL^(-1);建立的real-time PCR检测体系具有良好的线性关系,相关系数R2=0.9929,可特异性检测SdhB-H278Y突变,灵敏度为3.6×10^(-4) ng·μL^(-1),为AS-PCR的10倍。利用携带SdhB-H278Y突变不同比例的基因组DNA对检测体系进行验证,预期值与检测值具有很高的相关性,R^(2)=0.9997;利用该检测体系对山东地区黄瓜棒孢叶斑病病斑中多主棒孢SdhB-H278Y突变株所占比例进行检测,检测结果为0.12%~2.69%。综上,本试验建立的real-time PCR检测体系高效、灵敏、定量,可用于多主棒孢SdhB-H278Y突变的检测,为黄瓜棒孢叶斑病抗性治理提供技术支持。