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。

Prevalence and Antibiotic Resistance of Urinary Tract Pathogens, with Molecular Identification of Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp., Using Multiplex Real-Time PCR

Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.

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。

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。

PCR及Real-time PCR评价细菌DNA提取方法

目的:比较几种不同的细菌DNA提取方法,建立一种适于PCR和Real-time PCR的细菌DNA提取方法。方法:分别用蛋白酶K法、碱裂解法、Chelex-100+NP40、Chelex-100+TritonX-100法和水煮法提取同种浓度大肠杆菌DNA,测定OD260/280;用不同方法提取不同浓度的大肠杆菌DNA,进行PCR和实时荧光定量PCR扩增,比较灵敏度。结果:5种方法提取细菌DNA,其中Chelex-100+NP40法纯度(OD260/280=1.79±0.03)最高,此方法所提取的DNA产物进行PCR及Real-time PCR扩增未见假阳性及假阴性,其灵敏度为10个/ml细菌浓度。结论:Chelex-100+NP40的细菌DNA提取方法纯度及灵敏度高,且适应于PCR及Real-time PCR。

非洲猪瘟病毒常规PCR及Real-time PCR检测方法的建立

根据非洲猪瘟病毒(African swine fever virus,ASFV)P72基因的核苷酸序列,设计并合成引物以及荧光标记的TaqMan探针,以含P72基因的重组质粒作为阳性模板,用于常规PCR和Real-time PCR方法的建立,结果表明常规PCR的检测灵敏度是600个拷贝的病毒核酸分子,Real-time PCR的检测灵敏度是20个拷贝的病毒核酸分子,两种PCR检测方法均具有特异性强、简单快速的优点。可以用于出入境检验检疫部门对非洲猪瘟病毒的快速检测。

检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立

目的建立SYBR Green Real-time PCR检测和筛选黄病毒属病毒方法。方法参照文献报道的通用引物,以JEV cDNA和DEN cDNA为模板,建立RT-PCR和SYBR Green Real-time PCR,检测和筛选黄病毒属病毒,并比较两者的敏感性。结果此黄病毒属引物适合两种反应体系,SYBR Green Real-time PCR方法的敏感性是RT-PCR方法的100倍,最低检出病毒浓度为0.5×10-2PFU/ml。结论建立的两种方法均可用于黄病毒属病毒检测,以SYBR Green Real-time PCR方法具有更高的敏感性,对于黄病毒属病毒初筛检测具有应用价值。

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。

粪便标本溶组织内阿米巴原虫Real-Time PCR检测方法的建立及初步评价

为建立粪便标本溶组织内阿米巴原虫Real-Time PCR检测方法,并评价该方法在临床粪便标本检测中的应用价值,本文基于溶组织内阿米巴原虫小亚基核糖体RNA(SSU rRNA)基因序列设计了特异性引物并使用Real-Time PCR方法对溶组织内阿米巴原虫标准株DNA进行扩增,以建立溶组织内阿米巴原虫感染的Real-Time PCR技术。同时收集临床腹泻病人粪便标本221例并分别采用镜检法、Real-Time PCR法和ELISA原虫抗原检测法进行监测,以临床诊断结果为标准,分析3种方法的特异性、敏感性等检测性能。结果表明,本文建立的Real-Time PCR溶组织内阿米巴原虫检测方法特异性好,最低检测限为0.5 fg/μL,可用于临床腹泻患者粪便标本溶组织内阿米巴原虫检测。相对而言,Real-Time PCR方法特异性和敏感性均优于镜检法和ELISA原虫抗原检测法。

Development of TaqMan-based Real-time PCR Assay for Detecting Transmissible Gastroenteritis Virus and Its Application in Vaccine Evaluation

[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.

Establishment and Comparison of Two Taq Man Real-time PCR Methods for PCV2

[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was amplified in all the 8 samples, indicating the 8 samples were all infected with PCV2-1ike factor P1. [Conclusion] The ORFl-based detection method has a higher accuracy, and it can be used for the rapid detection of PCV2.

Real-Time PCR Technique and Its Application in Quantification of Plant Nucleic Acid Molecules

Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.

扇棘单睾吸虫Real-time PCR与常规PCR检测方法的建立

根据扇棘单睾吸虫核糖体DNA第一内转录间隔区(ITS1)序列,应用Primer premier 5.0软件自行设计一对可同时应用于real-time PCR和常规PCR的特异引物,建立real-time PCR与常规PCR鉴定扇棘单睾吸虫的方法,评估其特异性及灵敏性。应用建立的方法鉴定犬猫内脏中收集的吸虫,并用测序进行验证,检验方法的准确性和实用性。结果表明,建立的两种方法均只能特异性扩增扇棘单睾吸虫目的片段,不与横川后殖吸虫、钩棘单睾吸虫、华支睾吸虫、瓦氏瓦特松吸虫、棘口属吸虫、背孔属吸虫、心形咽口吸虫、野牛平腹盘吸虫、东方次睾吸虫、卫氏并殖吸虫、异尖属线虫、宫脂属线虫发生交叉扩增;敏感性试验表明,real-time PCR和常规PCR检测扇棘单睾吸虫质粒的最低检测限分别为43拷贝和86拷贝。建立的realtime PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(R2=0.998)。鉴定来自犬猫的吸虫17条,结果显示两种方法均能准确鉴定出扇棘单睾吸虫。

Establishment and Application of a Real-time PCR Method for Detecting stx2 Gene in Shiga Toxin-producing Escherichia coli(STEC)

[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples.

Advances in the Identification of Genetically Modified Rice with Real-time PCR and Multiplex PCR

In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.

病原真菌real-time PCR检测对非中性粒细胞缺乏的老年患者肺部真菌病的诊断价值

目的 评估本实验室设计的肺部常见病原真菌real-time PCR检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)标本对非中性粒细胞缺乏的老年人群肺部真菌病的诊断价值。方法 回顾性纳入2020年至2021年间于北京大学第一医院就诊的怀疑肺部感染的294名非中性粒细胞缺乏老年患者,采用real-time PCR检测其BALF标本中的病原真菌;根据宿主因素、临床表现和真菌学检查进行诊断分类,27名患者符合确诊或临床诊断的肺部侵袭真菌病,其中7例(25.6%)为多种真菌合并感染,6名患者诊断慢性肺曲霉病。结果 曲霉PCR Ct值受试者工作特征曲线下面积为0.936(95%置信区间0.865~1.000),最佳cut-off值为34.8,对肺曲霉病的诊断灵敏度和特异度分别为95.7%和92.1%。耶氏肺孢子菌PCR以Ct值37.1为cut-off值,诊断耶氏肺孢子菌肺炎灵敏度和特异度分别为94.7%和99.7%。隐球菌和毛霉PCR以Ct值35.0为cut-off值,在肺隐球菌病和肺毛霉病中阳性率分别为50%(2/4)和100%(1/1)。病原真菌real-time PCR确定cut-off值后,以PCR检出的真菌与肺部真菌病的致病真菌一致作为阳性,灵敏度、特异度、阳性预测值和阴性预测值分别为86.5%(32/37)、91.0%(253/278)、56.1%(32/57)和98.1%(253/258),总符合率为90.5%(285/315)。结论本研究团队开发的病原真菌real-time PCR体系覆盖常见肺部致病真菌,初步探索了其在BALF标本中的cut-off值,证实该方法在非中性粒细胞缺乏老年患者的肺部真菌病中具有良好的诊断价值。

寨卡病毒SYBR GreenⅠreal-time PCR方法的建立及应用

为建立一种针对寨卡病毒的快速诊断方法,本研究根据寨卡病毒的3’端保守基因序列,设计合成1对引物,建立了检测寨卡病毒的荧光定量PCR方法。结果显示:所建立的检测方法的Ct值与标准品在1.41×10^1~1.41×10^10^ copies/μL具有良好的线性关系,相关性为1,斜率为-3.502;灵敏性结果显示,该方法的检测限度为1.41×10^1 copies/μL,是普通PCR的10000倍;特异性结果显示,对CHIKV、DENV和JEV无特异性扩增,特异性强;重复性试验结果显示,组内和组间变异系数均小于1%,重复性好。本研究建立的SYBR Green I real-time PCR检测方法,可用于寨卡病毒感染的快速诊断。

Real-Time PCR检测棘阿米巴方法建立及应用

目的采用TaqMan探针法建立检测棘阿米巴18srDNA的实时荧光定量PCR（Real—timePCR）方法，为早期诊断棘阿米巴病提供基因诊断标准方法。方法提取实验室培养的4株土壤中分离的和1株水中分离的不同基因型棘阿米巴DNA，测序后在物种保守区域设计Real—time引物和TaqMan探针，用建立的方法检测动物模型兔眼分泌物、大肠杆菌、人巨细胞病毒、卡式肺孢子虫、疑似棘阿米巴角膜炎160例病人眼分泌物。结果与传统实验室培养病原相比，所建立的qPCR检测棘阿米巴角膜炎方法测定大肠杆菌、人巨细胞病毒和卡式肺孢子虫与该研究的实验室分离棘阿米巴物种没有交叉反应，检测结果均为阴性。而160例疑似病人，用培养法检测出感染棘阿米巴患者为5例，用qPCR方法检测出6例，其中5例为培养法测出患者，qPCR方法检测阳性率（3．75％）略高于普通培养法（3．13％），但无统计学意义（P〉O．05）。qPCR方法在95％置信区间中灵敏度100％（47．95％～100％）高于普通培养法83．33％（36．10％～97．24％）。结论所建立测定棘阿米巴18srDNA的Real-timePCR基因检测方法具有无创伤性、高效、特异、经济等特点，适用于疑似棘阿米巴角膜炎病人门诊筛选的有效方法。