添加扩增内标的沙门氏菌PCR检测方法

通过构建人工扩增内标,建立可以有效指示沙门氏菌检测过程可能出现假阴性情况的PCR检测方法。本研究基于沙门氏菌invA基因设计特异性引物,复合法构建扩增内标,建立PCR检测体系。特异性引物LW,对33株沙门氏菌和6株非沙门氏菌标准株进行检测,结果显示,所有沙门氏菌均扩增出385 bp的目标片段,非沙门氏菌则只能扩增出484 bp的扩增内标片段,特异性良好。灵敏度实验表明,该检测体系的灵敏度可达6.35 fg/μL。人工污染实验表明,起始染菌量为3.2 CFU/25 mL时,仅需8h增菌培养便可检出。大量食品样品检测证明,该检测体系确实可以有效的避免PCR检测过程出现的假阴性,提高检测准确性。     

扩增内标在沙门氏菌PCR检测方法中的应用

在PCR反应体系中添加了一条人工构建的扩增内标片段,以指示沙门氏菌PCR快速检测中出现的假阴性。对9株沙门氏菌和15株非沙门氏菌进行PCR检测,结果显示所有沙门氏菌都能扩增到一条invA基因中的374bp特异性片段,而模板来源于非沙门氏菌时则只能扩增到一条513bp扩增内标片段。灵敏度试验显示,该PCR检测体系对猪霍乱沙门氏菌纯DNA模板的检测灵敏度为12．8fg／μL,如果将增菌时间确定为8h,则该检测体系对人工染菌牛乳中沙门氏菌的检测灵敏度可以达到起始浓度为8cfu／25mL。采用上述方法检测了80份污染严重的样品,证实此方法可以有效地排除假阴性,提高检测准确率。     

扩增内标在沙门氏菌PCR检测方法中的应用*

在PCR反应体系中添加了一条人工构建的扩增内标片段,以指示沙门氏菌PCR快速检测中出现的假阴性。对9株沙门氏菌和15株非沙门氏菌进行PCR检测,结果显示所有沙门氏菌都能扩增到一条invA基因中的374bp特异性片段,而模板来源于非沙门氏菌时则只能扩增到一条513bp扩增内标片段。灵敏度试验显示,该PCR检测体系对猪霍乱沙门氏菌纯DNA模板的检测灵敏度为12·8fg/μL,如果将增菌时间确定为8h,则该检测体系对人工染菌牛乳中沙门氏菌的检测灵敏度可以达到起始浓度为8cfu/25mL。采用上述方法检测了80份     

添加有扩增内标的副溶血弧菌PCR检测方法的建立

摘要：【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌（Vibrio parahaemolyticus）基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物（vp1332L/vp1332R）,同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343 bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499 bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102 cfu/mL。人工污染实验表明,起始染菌量为1.24 cfu/25 g样品时经8 h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

添加有扩增内标的副溶血弧菌PCR检测方法

【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌(Vibrio parahaemolyticus)基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物(vp1332L/vp1332R),同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102cfu/mL。人工污染实验表明,起始染菌量为1.24cfu/25g样品时经8h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

抗草甘膦转基因大豆PCR检测及问题探讨

转基因植物的检测具有重要的意义。用抗草甘膦转基因大豆中的外源CaMV35S启动子、CP4 EPSPS和巢式PCR引物,应用PCR方法,从中扩增出预期大小的DNA片段。将扩增产物回收后测序,经同源性分析扩增产物为CaMV35S启动子和CP4 EPSPS的一部分序列。与常规PCR相比,巢式PCR在检测转基因大豆中具有更高的特异性。讨论了PCR检测过程中假阴性和假阳性的原因。     

添加有扩增内标的沙门氏菌荧光定量PCR 检测体系的建立与评价

【目的】建立添加有扩增内标(IAC,Internal amplification control)的沙门氏菌EvaGreen荧光定量PCR检测体系,提高PCR检测可靠性。【方法】通过比较已有沙门氏菌属细菌的基因组序列,筛选沙门氏菌属特异检测靶点,设计特异引物;再用复合引物法构建扩增内标,优化参数,建立沙门氏菌内标PCR检测体系,利用特异性和灵敏度实验评价体系的检测性能。【结果】筛选得到的新特异靶点基因编码III型分泌系统蛋白(ssaQ)。针对该基因设计特异引物(SsaQ6),建立了添加有扩增内标的常规PCR和EvaGreen荧光定量PCR检测体系;二者对151株沙门氏菌和34株非沙门氏菌的检测符合率均达100%,对基因组DNA的检测下限达14.9拷贝/PCR和2.76拷贝/PCR;人工污染牛奶样品(初始染菌量:4-6 cfu/10 mL),増菌10 h和8 h后分别可检出沙门氏菌。【结论】本研究发掘的新靶点基因ssaQ特异性强,基于这一新靶点建立的添加有扩增内标的EvaGreen荧光定量PCR比常规内标PCR的检测限更低,重复性更好,快速方便,在12 h内即可得出检测结果,并且定量准确,有利于推进沙门氏菌PCR检测方法的标准化应用。     

PCR防污染技术的研究进展

聚合酶链式反应（PCR）是一种高灵敏核酸扩增技术,广泛应用于核酸检测中。但在实际应用过程中,扩增产物及其他核酸片段的污染会导致假阳性的结果,制约了PCR在临床检测中的应用。为了解决这一问题,建立了许多PCR防污染的方法,除了早期建立的并已得到广泛应用的物理隔绝法、光照法及水解法外,近年来还发展了酶消化法、化学修饰法及DEAE纤维素法。本文对PCR防污染技术的原理、应用及进展进行了综述。     

分子信标-TaqMan探针法实时荧光定量PCR新型探针及引物

在TaqMan探针法实时荧光定量PCR中,排除由引物探针聚合延伸引起的假阳性问题以及由引物二聚体(Primer Dimer,PD)引起的假阴性问题。首先对不同探针进行引物探针聚合实验;然后通过双重PCR的干扰实验选择合适的内标基因,并使用内标质粒检测验证中部同序引物排除PD干扰的实用性;最后比较探针法不同体系检测HBV基因的灵敏度。结果表明,引物与探针聚合实验中,含有反义碱基的HBVP4在重复实验中未出现假阳性;竞争性双重PCR和非竞争性双重PCR两模板开始出现干扰作用的浓度差分别为20倍和100倍;对于内标基因,使用中部同序引物对以及普通引物对分别可以检测到10-9和10-8稀释度;3种HBV基因检测体系,使用普通引物对可以检测到Ct33左右,加入内标系统和使用中部同序引物对均可检测到Ct35左右。在TaqMan-分子信标结构调整的基础上引入反义碱基可以在排除由引物和探针聚合延伸引起的假阳性问题;在探针法中,使用中部同序引物对和加入内标系统均可降低PD的干扰程度,提升检测的灵敏度。     

PCR 防污染技术的研究进展

聚合酶链式反应(PCR)是一种高灵敏核酸扩增技术,广泛应用于核酸检测中.但在实际应用过程中,扩增产物及其他核酸片段的污染会导致假阳性的结果,制约了PCR在临床检测中的应用.为了解决这一问题,建立了许多PCR防污染的方法,除了早期建立的并已得到广泛应用的物理隔绝法、光照法及水解法外,近年来还发展了酶消化法、化学修饰法及DEAE纤维素法.本文对PCR防污染技术的原理、应用及进展进行了综述.     

Development of a multiple internal control for clinical diagnostic real-time amplification assays

A major pitfall in most published genomic amplification methods for the detection and identification of human pathogens is that they do not include an internal amplification control in order to achieve an acceptable level of confidence for the absence of false-negative results. By applying composite primer technology, a single multiple internal amplification control DNA molecule was constructed to detect and quantify the hepatitis B virus, human polyomavirus, Epstein-Barr virus, Toxoplasma gondii and cytomegalovirus using real-time PCR. The multiple internal amplification control contains all forward and reverse primer binding regions targeted in the five distinct duplex PCRs, but with a unique probe hybridization site. Multiple internal amplification control detection sensitivity, assessed by Probit analysis, was 58 copies per PCR, associated with an extremely wide dynamic range (8 log(10) units). Moreover, in testing 614 patient samples, PCR inhibition occurred at a frequency of 0-8.8%. Similar multiple internal amplification controls for quantitative PCR-based assays could be designed to accommodate any infectious profiles in a particular institution as they are easy to make and inexpensive.     

Selective PCR: a novel internal amplification control strategy for enhanced sensitivity in Salmonella diagnosis

Aims: The aim of this study was to develop a novel strategy that permits the independent amplification of internal amplification control (IAC) and target sequence using the same set of primers, to improve the sensitivity of diagnostic PCR assays. Methods and Results: The method described here is a Salmonella specific PCR test targeting the quorum sensing gene sdiA. It is based on a large size difference between the IAC and the target and consequently on their different extension time. The results indicate the enhanced sensitivity of this test when compared with the competitive IAC strategy. This is demonstrated through parallel testing of artificially contaminated human faecal samples. Conclusions: Utilizing this method, the concentration of the IAC, which often leads to false negative results if the target is present in extremely low concentration owing to competition, does not constitute a critical parameter for the detection limit of a PCR assay. Significance and Impact of the Study: To our knowledge, this is the first report of using extension time as a critical parameter for the sensitivity of a PCR test. A different approach for the construction of an IAC, based on inverse PCR, has also been introduced.     

Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood

A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V.parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 10⁷ CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.     

Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.     

Simple duplex fecal PCR assay that allows identification of false-negative results in Helicobacter sp.-infected mice

We designed a simple and sensitive duplex polymerase chain reaction (PCR) assay for detection of false-negative results during routine Helicobacter sp. feces analysis. We took advantage of the various Lactobacillus species that form part of the normal intestinal flora of laboratory rodents to improve our PCR diagnostic assays. Using this one-step PCR assay, we were able to rule out false-negative results without the need of adding internal standard molecules. This is an important quality control for PCR diagnostic tests, since the presence of inhibitors in feces can prevent detection of Helicobacter infections using PCR analysis. Use of this Lactobacillus group-specific PCR assay can be extended to other feces tests used in mouse quality-control programs.     

Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10(2) to at least 5 x 10(6) copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.     

Measurement of the efficacy of vaccines and antimicrobial therapy against infection with Toxoplasma gondii

To facilitate studies of vaccines and antimicrobial agents effective against Toxoplasma gondii infection, an assay system was developed to semi-quantitate parasitaemia using PCR amplification of T. gondii DNA obtained from the blood of mice infected with the parasite. A competitive internal standard DNA fragment of the B1 gene of T. gondii was generated and used in PCR so that the amplified product could be semi-quantitated and false negative results could be avoided. The PCR assay system was used to analyse the levels of parasitaemia in immunised and antimicrobial agent treated mice at various times after infection with T. gondii. The results of these studies indicate that this highly sensitive detection method is a rapid and reliable procedure that can be employed to assess the abilities of vaccines or antimicrobial agents to provide protection early following T. gondii infection.