A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

Species-specific DNA probe and development of a quantitative PCR assay for the detection of Morganella morganii

Aims: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning. Methods and Results: A new primer set, amplifying a 179‐bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non‐Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated. Conclusions: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples. Significance and Impact of the Study: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain.     

A quantitative assay for the detection of hepatitis B virus DNA employing a biotin-labeled DNA probe and the avidin-beta-galactosidase complex

We have developed a procedure for the quantitation of specific DNA which employs nonradioisotopic probes and beta-galactosidase as a detector. The sample DNA was immobilized on a nitrocellulose filter paper. After the filter paper had been processed to hybridization with a biotinylated probe DNA, the paper was incubated with avidin-beta-galactosidase complex. The optimum ratio of avidin to biotinylated beta-galactosidase for preparation of a complex between the two was determined. The filter paper was punched. Each punched piece was put into a microtiter well and beta-galactosidase activity was measured using 4-methylumbelliferyl beta-D-galactosidase as a substrate. By this method, we were able to quantify as little as a few picograms of specific DNA. The application of this method for the quantitative assay of hepatitis B virus DNA in serum sample is also described. The sensitivity for the detection of the DNA by our method was practically comparable to that of the conventional radioisotopic method. The validity of our method for detection of the virus DNA was further supported by comparison with the serological data.     

Biotinylated DNA probe for detection of streptotricin acetylation genes

The biotin-labelled DNA probe for identification of functioning and silent genes for streptotricin acetylation has been constructed. The probe is homologous to sat1 gene of the movable genetic element Tn1825. The simplified modification of the hybridization technique using the biotin-labelled DNA probe is described.     

A DNA probe for specific detection of Mycoplasma pulmonis

Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.     

基于TaqMan探针多重实时荧光PCR的肠道特殊菌群绝对定量方法的建立

本研究旨在建立对肠道主要共生菌的快速定量方法。根据细菌16S rRNA基因的保守序列并参考相关研究,设计针对总肠道菌群的通用引物和探针,以及针对双歧杆菌属、肠球菌属和肠杆菌科的特异性引物和探针,采用引物设计工具(Primer-BLAST) 以及聚合酶链式反应(polymerase chain reaction, PCR)扩增以验证引物和探针的特异性。通过分子克隆技术,构建各目标菌属目的基因的重组质粒作为qPCR检测的标准模板,选择标准模板进行重复性实验,并计算组内和组间重复变异系数。用所建立的方法对不同年龄段的临床粪便标本进行3类细菌的检测,并初步分析这些菌群与增龄的关系。结果显示,引物和探针具有较高的特异性;各目的细菌标准曲线在一定范围内线性关系良好,总菌群或各目标菌属的线性范围分别为：总菌群2.9×10³～2.9×10¹²copies/μL、双歧杆菌3.1×10²～3.1×10⁹ copies/μL、肠球菌5.9×10²～5.9×10⁹ copies/μL、肠杆菌6.3×10²～6.3×10⁹ copies/μL,决定系数R²≥ 0.995;检测的最低拷贝数为总菌群7.5×10² copies/μL、双歧杆菌 46 copies/μL、肠球菌 37 copies/μL、肠杆菌 51 copies/μL;重复性实验变异系数在1.32%以下,具有良好的可重复性。对不同年龄段临床粪便标本检测的结果显示,肠球菌和肠杆菌含量随增龄呈增长趋势,且老年组含量显著高于青年组,但在高龄老年人中未发现肠球菌数量增加。双歧杆菌含量随增龄减少,且各组间差异显著,在高龄老年人中也未观察到双歧杆菌显著减少。结果提示,本研究建立了一种可供临床推广的菌群绝对定量方法,能够同时检测3类细菌和菌群总量,对于菌群定量研究具有实际意义。     

TaqMan探针双重荧光定量PCR同时检测解脲支原体和巨细胞病毒

目的探讨双重荧光定量PCR技术的优化条件,建立基于TaqMan探针技术荧光定量法检测同时检测解脲支原体和巨细胞病毒的新方法。方法分别采用普通定性PCR扩增母婴垂直传播常见的病原体（解脲支原体和巨细胞病毒）测序鉴定,然后分别采用TaqMan探针的单重和双重定量PCR技术对解脲支原体和巨细胞病毒同时定性定量检测。结果解脲支原体和巨细胞病毒单种定性PCR检测均为阳性,TaqMan探针单重和双重定量PCR检测解脲支原体和巨细胞病毒阳性率和特异性均为100％,相同样品TaqMan探针单重、双重定量PCR分别检测的结果符合率100％。结论TaqMan探针双重荧光定量PCR技术可同时检测两种靶分子,结果可靠,应用前景广阔。     

Synthesis of a novel fluorescent probe useful for DNA detection

In this paper, a novel fluorescent probe 2-methylbenzo[b][1,10] phenanthrolin-7(12H)-one (m-BPO) is synthesized, and its molecular structure has been characterized by IR, UV, MS, (1)H-NMR and elements analysis. The fluorescent characteristics of m-BPO were investigated in detail. It was found that DNA had the ability to quench the fluorescence of m-BPO at 411 nm (lambda(ex)=286 nm), and the quenched intensity of fluorescence was proportional to the concentration of DNA. Based on this fact, m-BPO has been used as the fluorescent probe for detection of calf thymus DNA (ctDNA) and fish semen DNA (fsDNA). Under the optimal conditions, the calibration curves are linear up to 15.0 microg/ml for both ctDNA and fsDNA. The corresponding detection limits are 3.6 ng/ml for ctDNA and 5.5 ng/ml for fsDNA, respectively. The interaction mechanism for the binding of m-BPO to ctDNA was studied in detail, and the results suggested that the interaction mode between m-BPO and ctDNA was groove binding.     

Competitor internal standards for quantitative detection of mycoplasma DNA

Abstract Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumonias . To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.     

建立基于TaqMan探针的李坏死环斑病毒实时荧光RT-PCR检测方法

李坏死环斑病毒(Prunus necrotic ringspot virus, PNRSV)是世界部分范围内分布的有害生物, 亦是我国重点关注的检疫对象。根据PNRSV各株系衣壳蛋白基因的保守序列, 设计特异性引物和TaqMan荧光探针, 进行了探针、引物和Mg2+浓度等反应体系和条件的优化实验, 确定最佳的引物浓度为400 nmol/L、探针浓度为333 nmol/L、Mg2+离子浓度为5 mmol/L和dNTPs浓度为0.43 mmol/L时, 其灵敏度达23个拷贝数。利用建立的实时荧光RT-PCR检测方法对PNRSV樱桃分离物进行了成功检测。这个方法具有灵敏、准确、简便、快速的特点, 适合于李坏死环斑病毒的检测和鉴定。     

High capacity substrates as a platform for a DNA probe array genotyping assay

Colloidal silica particles were deposited on a glass substrate to produce high-capacity porous supports for high-density DNA probe arrays. Porous surfaces were used to increase the addressable surface area and number of probes available for hybridization. Surfaces derived from 70-100 nm size particles deposited in films from 0.15 to 2 microns thick exhibited excellent performance in light-directed oligonucleotide synthesis. Evaluation of these substrates in a genotyping assay is reported.     

Sensitive and specific DNA probe for detection of Plasmodium falciparum.

The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences.     

番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

【目的】本研究旨在建立TaqMan实时荧光定量PCR(TaqMan RT-qPCR)技术,快速检测单头烟粉虱Bemisia tabaci体内的番茄褪绿病毒(tomato chlorosis virus, ToCV)。【方法】根据ToCV外壳蛋白保守序列设计了1对特异性引物和1条TaqMan探针,建立了TaqMan RT-qPCR方法;与常规PCR检测进行比较,检测该方法的灵敏度与特异性;并应用该方法对单头烟粉虱成虫体内ToCV进行了快速检测。【结果】本研究构建的TaqMan RT-qPCR检测ToCV的标准曲线,其循环阈值(Ct值)与模板浓度具有良好的线性关系,扩增效率为98%。该方法对ToCV的最低检测浓度为8.3×10 copies/μL,灵敏度是常规RT-PCR的1 000倍。该方法与田间番茄两种重要病毒番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)和番茄斑萎病毒(tomato spotted wilt virus, TSWV)检测无交叉反应。单头烟粉虱成虫ToCV检测结果表明,温室内ToCV侵染植株上烟粉虱携毒率为100%,田间烟粉...     

Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence

Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis.     

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.     

基于TaqMan实时荧光定量PCR技术的西花蓟马快速检测

西花蓟马Frankliniella occidentalis（Pargande）是世界性害虫,2003年在我国首次发生危害。针对西花蓟马与其他种类蓟马形态相似、难以快速区分的问题,本文在SCAR标记基础上,采用TaqMan实时荧光定量PCR技术,设计1对特异性引物和1条MGB探针,扩增出大小为138bp的特异片段。以质粒DNA为标准品建立了标准曲线（R2=0.9965）,种特异性检验结果显示,该引物和探针只能检测到西花蓟马的荧光信号,而对其他种类的蓟马不具有检测能力。并且可以定量检测西花蓟马不同虫态靶标DNA片段的拷贝数。该检测体系重复性强、稳定性高,在口岸检疫以及植物种苗及其产品调运中的有害生物检测和监测中具有重要意义。     

TaqMan-MGB探针检测文森巴尔通体博格霍夫亚种实时荧光定量PCR方法的建立及应用

【目的】应用TaqMan-MGB探针技术,建立具有种水平特异性、高敏感性的荧光定量PCR方法,用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplifiedregion,SCAR)技术基础上,依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物,分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线,评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV值分别为0.12%-0.70%和0.14%-0.55%,在允许范围内;标准曲线线性关系良好(R2=1),扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

Use of TaqMan gene probe for real-time monitoring of acidophilic hydrogen-producing bacteria

Using a TaqMan gene probe targeting the 16S rDNA of acidophilic hydrogen-producing bacteria (HPB), the abundance of this HPB in the biomass was found to increase from 0.02 to 72% in a single batch treating rice slurry waste at pH 4.5 over 130 h. The corresponding abundances were 4.4% in the batch operated at pH 5.0 and 0.01–0.02% at pH 5.5–6.5. During the growth phase, the generation time for the acidophilic HPB at pH 4.5 averaged 3.5 h.