Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes

Aspergillus carbonarius is the main species responsible for ochratoxin A accumulation in wine grapes and consequently, its rapid and sensitive detection is increasingly investigated. A new real-time PCR (RTi-PCR) based procedure was developed for the rapid and specific detection and quantification of A. carbonarius in wine grapes. The procedure includes the use of the pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors, and DNA extraction with the EZNA Fungal DNA kit. It reduced the time for A. carbonarius DNA extraction from grapes to 30 min. Two specific primers (AcKS10L/AcKS10R) delimiting a 161 bp fragment, and a probe were designed and directed to the beta-ketosynthase domain of a polyketide synthase from A. carbonarius. Specificity was confirmed by testing primers towards purified DNA from 52 fungal strains, including reference and food isolates. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated conidial suspensions from A. carbonarius. The SYBR-Green I and TaqMan RTi-PCR approaches established were able to detect at least 2.4 and 24 genomic equivalents, respectively, using purified DNA. Results obtained from conidial suspensions, after DNA extraction, showed that at least 5 conidia per reaction should be present for a positive result with SYBR-Green I and 50 in the case of TaqMan. The quantification of fungal genomic DNA in artificially inoculated wine grapes performed successfully, with a minimum threshold of 10(3) conidia mL(-1) for accurate quantification. The developed RTi-PCR assay is a promising tool in the prediction of potential ochratoxigenic risk, even in the case of low-level infections, and suitable for a rapid, automated and high throughput analysis.     

Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR

Aspergillus carbonarius is an ochratoxin producing fungus that has been considered to be responsible of the ochratoxin A (OTA) contamination in grapes and wine. In order to monitor and quantify A. carbonarius, a specific primer pair Ac12RL_OTAF/Ac12RL_OTAR has been designed from the acyltransferase (AT) domain of the polyketide synthase sequence Ac12RL3 to amplify 141 bp PCR product. Among the mycotoxigenic fungi tested, only A. carbonarius gave a positive result. This specific primer pair was also successfully employed in real-time PCR conjugated with SYBR Green I dye for the direct quantification of this fungus in grape samples. A positive correlation (R(2)=0.81) was found between A. carbonarius DNA content and OTA concentration in 72 grape samples, allowing for the estimation of the potential risk from OTA contamination. Consequently, this work offers a quick alternative to conventional methods of OTA quantification and mycological detection and quantification of A. carbonarius in grapes.     

Development of a multiplex real-time PCR assay for the detection of ruminant DNA

The U.S. Food and Drug Administration (FDA) has previously validated a real-time PCR-based assay that is currently being used by the FDA and several state laboratories as the official screening method. Due to several shortcomings to the assay, a multiplex real-time PCR assay (MRTA) to detect three ruminant species (bovine, caprine, and ovine) was developed using a lyophilized bead design. The assay contained two primer or probe sets: a "ruminant" set to detect bovine-, caprine-, and ovine-derived materials and a second set to serve as an internal PCR control, formatted using a lyophilized bead design. Performance of the assay was evaluated against stringent acceptance criteria developed by the FDA's Center for Veterinary Medicine's Office of Research. The MRTA for the detection of ruminant DNA passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements by detecting 30 of 30 complete feed samples fortified with meals at 0.1 % (wt/wt) rendered material from each of the three ruminant species. The MRTA demonstrated 100 % selectivity (0.0 % false positives) for negative controls throughout the assessment period. The assay showed ruggedness in both sample selection and reagent preparation. Second and third analyst trials confirmed the quality of the written standard operating procedure with consistency of results. An external laboratory participating in a peer-verification trial demonstrated 100 % specificity in identifying bovine meat and bone meal, while exhibiting a 0.03 % rate of false positives. The assay demonstrated equal levels of sensitivity and reproducibility compared with the FDA's current validated real-time PCR assay. The assay detected three prohibited species in less than 1.5 h of total assay time, a significant improvement over the current real-time assay. These results demonstrated this assay's suitability for routine regulatory use both as a primary screening tool and as a confirmatory test.     

Development of a real-time PCR assay for the detection of cow and donkey milk

A real-time PCR allelic discrimination TaqMan assay based on the analysis of a single nucleotide polymorphism enabling the differentiation of cow (Bos taurus) and donkey (Equus asinus) milk was developed. Specific primers and probes were designed on the mitochondrial cytochrome c oxidase subunit I gene. The primers were designed upstream and downstream the chosen diagnosis site in a conserved region. Two probes were designed to specifically hybridise to B. taurus and E. asinus sequences. The test allowed the discrimination of bovine and donkey DNA in all blood and pure milk samples giving an unambiguous result plot of rapid and easy interpretation. The detection threshold was 2?% of cow milk in donkey milk. The applicability of the method to matrices containing degraded DNA was demonstrated by analysing samples of raw donkey and cow milk autoclave-treated (121?°C for 15?min). Finally, the assay when applied to milk samples collected from the retail trade has confirmed the species indicated in the label. Furthermore, the assay represents a potentially valuable diagnostic tool for species identification in dairy products for allergic people.     

Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 10⁴ conidia due to higher frequency of resistance colonies (894 per 10⁴ conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries.     

Early detection of Aspergillus carbonarius and A. niger on table grapes: a tool for quality improvement

Aspergillus carbonarius and A. niger aggregate are the main fungal contaminants of table grapes. Besides their ability to cause black rot, they can produce ochratoxin A (OTA), a mycotoxin that has attracted increasing attention worldwide. The objective of this work was to set up a simple and rapid molecular method for the early detection of both fungi in table grapes before fungal development becomes evident. Polymerase chain reaction (PCR)-based assays were developed by designing species-specific primers based on the polyketide synthases (PKS_S) sequences of A. carbonarius and A. niger that have recently been demonstrated to be involved in OTA biosynthesis. Three table grape varieties (Red globe, Crimson seedless, and Italia) were inoculated with A. carbonarius and A. niger aggregate strains producing OTA. The extracted DNA from control (non-inoculated) and inoculated grapes was amplified by PCR using ACPKS2F-ACPKS2R for A. carbonarius and ANPKS5-ANPKS6 for A. niger aggregate. Both primers allowed a clear detection, even in symptomless samples. PCR-based methods are considered to be a good alternative to traditional diagnostic means for the early detection of fungi in complex matrix for their high specificity and sensitivity. The results obtained could be useful for the definition of a ‘quality label’ for tested grapes to improve the safety measures taken to guarantee the production of fresh table grapes.     

Modelling the limit of detection in real-time quantitative PCR

The limit of detection (LOD) is a critical performance characteristic of an assay that requires careful evaluation during method validation. However, formal calculations for the LOD do not take into account atypical data sets that are generated from real-time PCR techniques, which can be non-normally distributed, truncated, and heteroscedastic. Experimental data sets for the quantification of genetically modified (GM) material were produced using real-time PCR, in order to model the LOD. A bootstrapping computer simulation calculated the probabilities of detecting PCR positive test results from these data sets, and computer modelling defined a function from the resulting probability plots. The LOD was modelled as a function of sample replication level and cycle threshold values. The broad applicability of this bootstrapping and data modelling approach should be of general interest to laboratories conducting trace-level detection.     

Survival and growth of Aspergillus carbonarius on wine grapes before harvest

Aspergillus carbonarius, the primary OTA-producing species in Australia, was inoculated onto the surface of Chardonnay and Shiraz bunches at pre-bunch closure, veraison and pre-harvest during the 2002-03 and 2003-04 seasons. Mean A. carbonarius counts decreased between pre-bunch closure and veraison, and increased between veraison and pre-harvest. Increases in A. carbonarius counts from veraison onwards were most marked in Chardonnay bunches during 2003-04; such bunches comprised more berries and were heavier than in 2002-03. Bunches with no berry damage yielded low A. carbonarius counts at pre-harvest and harvest. Exposure to direct sunlight over several days reduced viability of A. carbonarius spores supported on filter membranes by 10(5), despite the spores having thick, heavily melanised walls. The estimated cumulative UV exposure for that period was 10 mWh. Thus, UV radiation may be a contributory factor to the decline of A. carbonarius spores on berry surfaces, particularly in the early stages of berry development.     

Development of a new real-time quantitative PCR assay for the detection of Staphylococcus aureus genotype B in cow milk,targeting the new gene adlb

The specific and reliable diagnosis of mastitis pathogens is essential for successful sanitation programs. The aim of the present study was to develop and evaluate a new real-time quantitative PCR (qPCR) assay for the very sensitive and specific detection of Staphylococcus aureus genotype B in cow milk samples. This mastitis pathogen is contagious and particularly prevalent in Switzerland and other central European countries. The new test is based on a rapid preparation of bacteria, followed by DNA isolation and qPCR for a unique target gene coding for the adhesion-like bovine protein (adlb). The inclusivity of the new target gene was 97% and the exclusivity 98%, meaning that other genotypes and bacterial species could be excluded with high reliability. The limit of detection of the new assay was 235 staphylococcal cell equivalents/mL of culture. The new test shows high intra- and interassay repeatability. Results are available within 2 d after sampling, allowing farmers and veterinarians to apply sanitation measures immediately. Based on the results of a preliminary field study, the diagnostic sensitivity and specificity of the new qPCR assay are 99 and 100%, respectively. The new analytical procedure is straightforward and can be applied for routine diagnostics.     

基于内参系统的阪崎肠杆菌实时荧光定量PCR检测方法的建立

根据阪崎肠杆菌ompA靶基因设计特异性引物和探针,并加入内参(IAC),建立能够实时监控反应过程的荧光定量PCR检测方法。结果表明,该方法对阪崎肠杆菌基因组DNA的最低检测限为1pg;对细菌的最低检测限为1×10~4 CFU;对含有靶基因质粒的最低检测限为10~3拷贝;Ct值与模板拷贝数均呈良好的线性关系(R~2=0.999)。人工污染试验结果表明,在初始菌量为10CFU/25g奶粉样品时,采用水洗加试剂盒法和水煮法提取DNA,阪崎肠杆菌均在增菌10h时检出。研究结果为进一步优化和完善阪崎肠杆菌分子生物学方法的标准化提供了参考。     

实时荧光定量PCR在食品检测领域中应用

实时荧光定量PCR是在定性PCR技术基础上发展起来的核酸定量技术;该技术不仅实现对DNA模板定量,且具有灵敏度高、特异性强、无污染性、及实时性和准确性等特点。该文主要介绍实时荧光定量PCR原理和在食品检测中应用,及其在食品领域发展前景。     

应用多重实时荧光PCR检测蜡样芽胞杆菌毒力基因

目的:建立一种快速、准确、简便的蜡样芽胞杆菌毒力基因检测方法,为蜡样芽胞杆菌更深入的研究提供依据和便利。方法:采用TaqMan探针法设计了三重hblA,hblC,hblD基因,三重nheA,nheB,nheC基因,三重ces,entFM,bceT基因的多重实时荧光PCR体系检测蜡样芽胞杆菌毒力基因。结果:多重体系对蜡样芽胞杆菌毒力基因的检测具有良好的特异性和灵敏性,其最低检出限为63 CFU/mL。结论:利用上述体系对蜡样芽胞杆菌的毒力基因进行检测能够省时省力,特异性、灵敏性良好,具有实际应用价值。     

肉制品中鸭源性成分TaqMan探针实时荧光PCR检测方法的建立与应用

目的 建立TaqMan探针实时荧光PCR法快速检测肉制品中鸭源性成分的分析方法.方法 以鸭生长激素(growth hormone,GH)基因为靶基因,基于特异性保守序列设计引物和TaqMan探针,优化反应体系和反应条件,建立了鸭源性成分实时荧光PCR(real-time PCR)检测方法.结果 所建立的real-tim...     

Towards a quantitative application of real-time PCR technique for fish DNA detection in feedstuffs

A real-time PCR method to detect fish DNA in feedstuffs was developed and optimised. A combination of primers and a Taqman-MGB probe was used to selectively amplify the fish mitochondrial 12S ribosomal RNA gene. Qualitative and also quantitative assessments were performed with different protocols: a relative quantification by a standard curve, and a ΔC_T method, by total plant DNA as endogenous controls. Method specificity was evaluated analysing 40 different tissues (mammalians, avian, fish) and flour samples. Sensitivity was evaluated by LOD (limit of detection) estimation. The designed probe–primers set showed an increased sensitivity compared to previously published PCR end point method, reaching a limit of detection of 0.2 pg of fish DNA, and showing to be a robust assay for fish DNA detection. The quantification results, based on ΔC_T method and the relative standard curve, are well reproducible in our experimental condition but, in lacking of separate pure raw materials of a tested feed, they cannot be applied for reliable and precise quantification on field samples but for now as a semi-quantitative PCR method only.     

TaqMan实时荧光聚合酶链式反应(PCR)技术定量检测婴幼儿配方食品中的金黄色葡萄球菌

靶向于耐热核酸酶基因nuc,研究建立了Taq Man实时荧光聚合酶链式反应(PCR)(qRT-PCR)法用于婴幼儿米粉及乳粉中金黄色葡萄球菌的快速定量检测。经优化的qRT-PCR体系特异性强,仅在金黄色葡萄球菌中产生典型扩增曲线;方法灵敏度高,对标准质粒、纯菌液、模拟染菌米粉及奶粉样液的检测限分别为17.94拷贝、1.2 CFU、0.42 CFU和0.74 CFU/PCR反应体系;10倍系列梯度稀释的标准质粒、纯菌及人工染菌样液的浓度对数与qRT-PCR的Ct值线性相关度良好,拟合方程的决定系数均≥0.99;平板计数法与qRT-PCR法对模拟米粉及乳粉盲样中金黄色葡萄球菌的定量检测结果间无统计学差异。文中所建立的qRT-PCR定量法特异性强、灵敏度高,简便快捷、可靠性佳,可为婴幼儿米粉及乳粉中金黄色葡萄球菌的快速、准确定量检测提供有效手段。     

Development of a PCR assay for detection of Enterobacteriaceae in foods

A broad-range PCR assay for the detection of bacteria belonging to the Enterobacteriaceae family was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 72 different bacterial species (of 49 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Vibrioaceae or Enterobacteriaceae strain except Proteus mirabilis. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA for PCR was prepared by a simple procedure with the use of Chelex 100 resin from culture after growth in brain heart infusion medium. To test this PCR assay for the monitoring of the Enterobacteriaceae family, either Escherichia coli or Salmonella Enteritidis was inoculated into various foods as an indicator. Prior to the PCR, the inoculation of 10 to 40 CFU of bacteria per g of food was followed by a 5-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. When actual examinations of the contamination of 15 noodle foods with Enterobacteriaceae by this PCR assay were conducted, 33% (5 of 15) of the samples tested positive. These results agreed with those of the Petrifilm Enterobacteriaceae Count Plate assay. Including the enrichment culture step, the entire PCR detection process can be completed within 7 h.     

实时荧光定量技术在食品微生物检测和研究中的应用

实时荧光定量PCR技术是通过检测PCR产物中荧光讯号强度来达到定量的目的,不仅实现了对核酸信息量的分析比较,而且与常规PCR相比,它具有特异性更强、能有效解决PCR污染问题、自动化程度高等特点。文章概述了实时荧光定量PCR技术的原理、优缺点及其在食品微生物检测中的应用与研究进展,并探讨了它的技术发展和应用前景。     

实时荧光定量PCR技术快速检测志贺氏菌

目的 建立实时荧光定量PCR法快速检测志贺氏菌。方法 提取志贺氏菌基因组为模板扩增virA基因片段, 将目的片段连接至pMD19-T载体得到重组质粒, 转化大肠杆菌DH5α感受态细胞, 验证所得到的重组质粒, 以紫外分光光度计测量重组质粒吸光度值A260, 并换算为质粒拷贝数后作梯度稀释得到不同浓度的质粒标准品; 进行荧光定量PCR分析并通过特异性、灵敏性实验以验证该方法的可行性。结果 重组质粒标准品浓度在103~108 copies/μL范围内线性关系良好(r2>0.99), 实时荧光定量PCR检测志贺氏菌时出现良好的扩增曲线, 鼠伤寒沙门氏菌、金黄色葡萄球菌和大肠杆菌均未出现扩增曲线。志贺氏菌的检出限为100 CFU/mL。 结论 本方法便捷、高效、可靠, 可用于志贺氏菌的快速定性定量检测。     

Development of a real-time PCR assay with an internal amplification control for detection of Gram-negative histamine-producing bacteria in fish

Prompt detection of bacteria that contribute to scombrotoxin (histamine) fish poisoning can aid in the detection of potentially toxic fish products and prevent the occurrence of illness. We report development of the first real-time PCR method for rapid detection of Gram-negative histamine-producing bacteria (HPB) in fish. The real-time PCR assay was 100% inclusive for detecting high-histamine producing isolates and did not detect any of the low- or non-histamine producing isolates. The efficiency of the assay with/without internal amplification control ranged from 96-104% and in the presence of background flora and inhibitory matrices was 92/100% and 73-96%, respectively. This assay was used to detect HPB from naturally contaminated yellowfin tuna, bluefish, and false albacore samples. Photobacterium damselae (8), Plesiomonas shigelloides (2), Shewanella sp. (1), and Morganella morganii (1) were subsequently isolated from the real-time PCR positive fish samples. These results indicate that the real-time PCR assay developed in this study is a rapid and sensitive method for detecting high-HPB. The assay may be adapted for quantification of HPB, either directly or with an MPN-PCR method.     

驼乳中布鲁氏菌实时荧光PCR快速检测方法的建立

目的 建立驼乳中布鲁氏菌的实时荧光PCR快速检测方法。方法 根据布鲁氏菌外膜蛋白omp10基因的碱基序列,设计引物其探针,从模拟布鲁氏菌污染的驼乳中提取布鲁氏菌DNA片段进行实时荧光PCR扩增检测。结果 该方法从布鲁氏菌M5株中扩增出了特异性目的片段omp10,而对大肠埃希式杆菌等细菌的扩增结果均为阴性。经过10倍倍比稀释的模板进行检测,该方法的灵敏度为最低可检出2.31×10-4 ng/μL的DNA。标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.9928,扩增效率E=0.97。结论 建立的实时荧光PCR检测方法具有特异性强、灵敏度高的优点,为驼乳中布鲁氏菌病的早期预防及流行病学监测提供了有效的技术手段。