聚合酶链反应在研究DNA多态性中的应用

聚合酶链反应(Polymerase chain reaction PCR)是一种体外DNA扩增技术,自1985年Saiki等首次报道PCR方法以来,PCR技术迅速发展,并已广泛应用于生命科学各个方面。如基因克隆、遗传病的诊断、传染病的诊断、癌基因的检测等,由于PCR技术特异性强,扩增效率高、快速、录敏,且能克服限制性片段长度多态性(RFLP)的局限性,因此,它被有效地应用于研究DNA多态性。     

扩增大片段核酸的聚合酶链反应及其应用

本综述了扩增１０－４０ｋｂ大片段核酸的聚合酶链反应方法的原理,影响因素条件优化,以及ＬＡ－ＰＣＲ在扩增与克隆目的的基因、研究基因组的多态性和分别等方面的应用。     

聚合酶链反应在牛血清支原体检测中的应用

为了探讨聚合酶链反应在牛血清支原体检测上的应用价值,以支原体高度保守的rRNA操纵子(支原体基因组中16SrRNA的编码区序列)设计引物,采用碱裂解法提取牛血清中支原体DNA作为模板进行聚合酶链反应。结果表明,阳性、阴性和内控对照都扩增出了预期的条带,聚合酶链反应与支原体培养法比较,有灵敏、快速、特异性高的特点,可用于牛血清中支原体的常规检测。     

聚合酶链反应技术在脑脊液病毒学诊断中的应用

近年来中枢神经系统病毒感染性疾病发病率上升,确诊的主要依据是脑脊液的病原学诊断,但脑认中病毒含量甚低,其病毒培养检测不但费时,费力,而且敏感性和特异性很差,远远不能满足临床要求,建立在聚合酶链反应技术基础上的各种分子生物学诊断技术,逐渐成为生物医学领域最有价值的研究手段和病毒学诊断方法,已经越来越多地应用于中枢神经系统病毒学的基因诊断,本文对近几年聚合酶链反应技术的研究进展进行了综述。     

套式聚合酶链反应在HIV-1检测中的应用

根据HIV-1 gag.pol和env基因序列设计了套式聚合酶链反应(nesled polymerasechain reaction,nPCR)引物。将外周血单个核细胞裂解液先用外侧引物扩增,其产物再经内侧引物扩增,直接走凝胶电泳判定结果。nPCR的敏感性。较常规PCR高100～1000倍,可检测到0.5fg质粒DNA和50μ1HIV-1感染者外周血样中的HIV基因。用本法检测63名HIV-1感染者全为阳性,而61名健康人和可疑者均为阴性,后者经追踪检测抗体排除了HIV-1感染。实验证明,nPCR是一种敏感性极高、特异性很强、操作简便易行的HIV-1基因检测技术。它已经在早期诊断和外周血病毒含量检测中发挥了重要作用,而且有着更广阔的应用前景。     

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Diagnosis of bovine freemartinism by the polymerase chain reaction method

Summary. Using sex chromosome specific primers, leucocyte chimaerism in heterosexual bovine female twins was identified by combining polymerase chain reaction (PCR) and restriction enzyme digests.     

Miniaturization of polymerase chain reaction

Polymerase chain reaction (PCR) is one of the most widely used analytical tool and is an important module that would benefit from being miniaturized and integrated onto diagnostic or analytical chips. There are potentially two different approaches for the miniaturization of the PCR module: chamber-type and flow-type micro-PCR. These miniaturized PCRs have distinct characteristics and advantages. In this article, we review the necessity of micro-PCR, the materials for the chip fabrication, the surface modification, and characteristics of the two types of micro-PCR. The motivation underlying the development of micro-PCR, the advantages and disadvantages of the various materials used in fabrication and the surface modification methods will be discussed. And finally, the precise features of the two different types of micro-PCR will be compared.     

Detection and identification ofCampylobacter coli andCampylobacter jejuni by two-step polymerase chain reaction

Flagellin gene was used as target sequence to detect and distinguishC. coli andC. jejuni by a “nested PCR” technique. The method shows a high level of sensitivity and specificity. Application of this rapid diagnostic tool could provide further information about epidemiological and pathogenetic implications of each of these two microorganisms.     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR). The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups. AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.     

Rapid identification of Triticeae genotypes from single seeds using the polymerase chain reaction

An easy and quick protocol has been developed for DNA analysis via PCR. Single cereal endosperm or small leaf pieces can be separately processed in several PCR reactions. The resultant PCR patterns are equivalents to those obtained with standard DNA extraction protocols using either specific or random primers. Intra-and inter-specific variability can be detected. This method allows the analysis of a large number of individuals in early stages prior to the plant sowing.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.