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1000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%,田间烟粉虱的携毒率为30%。【结论】本研究建立的TaqMan RT-qPCR检测方法,可快速有效检测单头烟粉虱体内ToCV携毒情况,为该病毒病的防控提供了技术支撑。     

A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.     

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus

Xanthophylls are oxygenated carotenoids that perform critical roles in plants. -carotene hydroxylases (-hydroxylases) add hydroxyl groups to the -rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis. The lut1 mutation of Arabidopsis disrupts -ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on -ring structures. We have used library screening and genomics-based approaches to isolate a second -hydroxylase genomic clone and its corresponding cDNA from Arabidopsis. The encoded protein is 70% identical to the previously reported Arabidopsis -hydroxylase 1. Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent. Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, -hydroxylase 1 mRNA is always present at higher levels. Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of -ring containing carotenes and are only weakly active towards -ring containing carotenes. Neither -hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase. These data indicate that the LUT1 locus encodes a protein necessary for -ring hydroxylation but unrelated to -hydroxylases at the level of amino acid sequence.     

Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5'-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards

This study describes a multiplex real-time polymerase chain reaction (PCR) assay that quantifies total mitochondrial DNA (mtDNA(total)) and mtDNA bearing the 4977-base pair 'common deletion' (deltamtDNA4977) in lymphoblasts derived from an individual diagnosed with Pearson's syndrome. The method is unique in its use of plasmids as external quantification standards and its use of multiplex conditions. Standards are validated by comparison with purified mtDNA amplification curves and by the fact that curves are largely unaffected by nuclear DNA (nucDNA). Finally, slopes of standard curves and unknowns are shown to be similar to each other and to theoretical predictions. From these data, mtDNA(total) in these cells is calculated to be 3258 (+723/-592) copies per cell while deltamtDNA4977 averages 232 (+136/-86) copies per cell or 7% (+4.65/-2.81).     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

A species-specific real-time PCR assay for identification of three lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica

We present a multiplex real-time PCR assay for the simultaneous identification of three morphologically similar species of lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica. Based on TaqMan MGB (minor groove binding) probes targeting the fungal internal transcribed spacer (ITS nrDNA) region, our assay unambiguously identifies known samples from all the three species, thus providing a powerful alternative to the more expensive DNA-sequencing techniques.