转基因植物Real-time PCR方法检出限和定量限的研究

目前Real-timePCR(Rt-PCR)方法在转基因植物定量检测中应用最为广泛。有关该方法的检出限和定量限的计算并没有达成统一。本文采用统计模型对三种转基因植物进行实时荧光定量PCR方法检出限和定量限的研究。实验结果表明:转基因玉米NK603检出限5拷贝,定量限14拷贝;转基因棉花Mon15985检出限5拷贝,定量限12拷贝;转基因油菜Oxy235检出限4拷贝,定量限9拷贝。是利用统计学方法对转基因植物荧光定量PCR方法检出限和定量限研究的一个积极尝试,可用于其他转基因植物检测中检出限和定量限的确定。     

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography–mass spectrometry (HPLC–MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products.     

番茄交链孢病原菌鉴定及产毒分析

目的 通过对番茄病果中链格孢病原菌的分离鉴定及产毒分析,为番茄采收后病害防控提供理论依据。方法 采集福建省5个县市番茄病果,通过切片和组织分离法从番茄病害部位分离纯化病原菌,结合形态学特征和分子生物学对病原菌进行鉴定。根据柯赫氏法则确定病原菌的致病性,并利用超高效液相串联质谱法对分离得到的交链格孢菌产毒能力进行检测分析。结果 在发病番茄病害部位共分离得到的 3株互隔链格孢菌株均具产毒能力,但毒素种类和产毒能力各有不同,其中NH06菌株产毒量最高,可产生细交链格孢酮酸(tenuazonic acid,TeA )、交链孢酚 (alternariol, AOH)、交链孢酚单甲醚(alternariol monomethyl ether, AME)、交链孢烯(altenuene,ALT )、腾毒素 (tentoxin,TEN ) 5种毒素,而NP002和NH07仅检出产生4种毒素。结论 福建省番茄种植产区内存在交链格孢菌的污染,且产毒种类多,因此,在番茄种植产区内病果的处理应引起关注和重视。     

Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant

Real-time PCR assays using TaqMan or Molecular Beacon probes were developed and optimized for the quantification of total bacteria, the nitrite-oxidizing bacteria Nitrospira, and Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) in mixed liquor suspended solids (MLSS) from a municipal wastewater treatment plant (WWTP) using a single-sludge nitrification process. The targets for the real-time PCR assays were the 16S rRNA genes (16S rDNA) for bacteria and Nitrospira spp. and the amoA gene for N. oligotropha. A previously reported assay for AOB 16S rDNA was also tested for its application to activated sludge. The Nitrospira 16S rDNA, AOB 16S rDNA, and N. oligotropha-like amoA assays were log-linear over 6 orders of magnitude and the bacterial 16S rDNA real-time PCR assay was log-linear over 4 orders of magnitude with DNA standards. When these real-time PCR assays were applied to DNA extracted from MLSS, dilution of the DNA extracts was necessary to prevent PCR inhibition. The optimal DNA dilution range was broad for the bacterial 16S rDNA (1000-fold) and Nitrospira 16S rDNA assays (2500-fold) but narrow for the AOB 16S rDNA assay (10-fold) and N. oligotropha-like amoA real-time PCR assay (5-fold). In twelve MLSS samples collected over one year, mean cell per L values were 4.3 +/- 2.0 x 10(11) for bacteria, 3.7 +/- 3.2 x 10(10) for Nitrospira, 1.2 +/- 0.9 x 10(10) for all AOB, and 7.5 +/- 6.0 x 10(9) for N. oligotropha-like AOB. The percent of the nitrifying population was 1.7% N. oligotropha-like AOB based on the N. oligotropha amoA assay, 2.9% total AOB based on the AOB 16S rDNA assay, and 8.6% nitrite-oxidizing bacteria based on the Nitrospira 16S rDNA assay. Ammonia-oxidizing bacteria in the wastewater treatment plant were estimated to oxidize 7.7 +/- 6.8 fmol/hr/cell based on the AOB 16S rDNA assay and 12.4 +/- 7.3 fmol/hr/cell based on the N. oligotropha amoA assay.     

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.     

ITS-based detection and quantification of Alternaria spp. in raw and processed vegetables by real-time quantitative PCR

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of Alternaria spp. in foodstuffs. The method uses Alternaria-specific primers and probe targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 190 commercial food samples, including 80 fresh fruit and vegetable samples and 110 processed foodstuffs. The assay demonstrated the presence of Alternaria spp. DNA in 46 out of the 80 raw samples (57.5%) and in 66 out of the 110 processed samples (60%), enabling quantitative detection of Alternaria spp. DNA at levels as low as 1 CFU/g. The estimated Alternaria counts obtained by real-time PCR showed a good relationship (R² = 0.9006, P < 0.01) with the Alternaria counts obtained by plating on Potato Carrot Agar (PCA). The developed real-time PCR assay provides a useful tool for early detection of Alternaria spp. and could be applied as a quality and biosecurity marker of raw materials and final products in the fruits and vegetables processing industries.     

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.     

Real-time PCR assays for strain-specific quantification of probiotic strains in human faecal samples

Strain-specific real-time PCR assays were developed for the probiotic strains Bifidobacterium breve 99, Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii ssp. shermanii JS, used in combination, for quantification of the strains in human faecal samples. Unique randomly amplified polymorphic DNA (RAPD) sequences or a phage-related sequence of the strains were used as a basis for designing strain-specific primers and probes. The real-time PCR conditions were optimised and the specificities of the assays were confirmed by analysing DNA from related bacterial strains and from faecal samples originating from an intervention study. The assays allowed quantification with a linear range of six orders of magnitude, from 10¹ to 10⁷ target genomes per assay. The detection limit was 4 × 10⁵ genome copies per g of faeces. The strain-specific quantitative real-time PCR assays developed were suitable for identification and quantification of probiotic strains in human faecal samples in a controlled intervention trial.     

Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group

Five different enterotoxins and one emetic toxin of Bacillus cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex PCR with 12 primer pairs was established. In developing the assay method, a common terminal sequence at the 3' ends of all primers was chosen and a hot start Taq polymerase was used to overcome primer dimer formation. The assay was successfully applied to analyze the toxigenic potential of 162 food-poisoning and food-related strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of Bacillus mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains (P < 0.05), although the two species were closely related. The results suggest that many B. mycoides strains might be less prone to cause food poisoning. They also indicate the importance of detecting the toxin genes together with the detection of the species in the B. cereus group.     

Real-time PCR multiplex method for the quantification of Roundup Ready soybean in raw material and processed food

This paper describes a quantitative real-time multiplex PCR method optimised for the ABI PRISM^® 7700 Sequence Detection System (SDS) and TaqMan^® chemistry for Roundup Ready^® Soybean (RRS) in raw material and processed food. This method has the advantage of performing the amplification of the target taxon lectin gene and the genetically modified (GM) target sequence in the same test tube. The quantification is based on a calibration curve obtained with the DNA extracted from Certified Reference Material standards (CRM IRMM-410: R) in the range 0.1–5% RRS. The method was validated in-house by using CRMs and the applicability was verified on three mixtures of soybean flour at 1, 5, 10% of RRS respectively and on prepared baked products (biscuits and plum cakes). The statistical parameters of the method were found to be satisfactory both for soybean flour and baked products. In the process the absolute LOD and LOQ was 7.1 and 35.5 copy number respectively; the relative LOD and LOQ was 0.03 and 0.01% of RRS respectively.     

运用Real-time quantification PCR方法建立副溶血性弧菌在即食虾中的生长预测模型

运用Real-time quantification PCR(real-time qPCR)方法建立副溶血性弧菌在即食虾中生长预测模型.首先构建质粒标准品,梯度稀释后建立标准曲线,然后用Real-time qPCR方法检测虾中副溶血性弧菌的数量,最后建立37℃下即食虾中副溶血性弧菌生长预测模型,并与传统涂布计数方法进行比较.结果表明,Real-time qPCR方法和传统计数方法均可建立Gmopertz模型、Logistic模型和Richards模型,模型拟合的相关系数R2均在0.9以上.基于Real-time qPCR方法省时省力、特异性好等优点,用Real-time qPCR方法建立微生物预测模型是未来预测微生物学领域的一种发展趋势.     

运用Real-time quantification PCR方法建立副溶血性弧菌在即食虾中的生长预测模型

运用Real-time quantification PCR（real-time qPCR）方法建立副溶血性弧菌在即食虾中生长预测模型。首先构建质粒标准品,梯度稀释后建立标准曲线,然后用Real-time qPCR方法检测虾中副溶血性弧菌的数量,最后建立37℃下即食虾中副溶血性弧菌生长预测模型,并与传统涂布计数方法进行比较。结果表明,Real-time qPCR方法和传统计数方法均可建立Gmopertz模型、Logistic模型和Richards模型,模型拟合的相关系数R2均在0.9以上。基于Real-timeqPCR方法省时省力、特异性好等优点,用Real-time qPCR方法建立微生物预测模型是未来预测微生物学领域的一种发展趋势。      

PCR amplification of repetitive sequences as a possible approach in relative species quantification

Both relative and absolute quantifications are possible in species quantification when single copy genomic DNA is used. However, amplification of single copy genomic DNA does not allow a limit of detection as low as one obtained from amplification of repetitive sequences. Amplification of repetitive sequences is therefore frequently used in absolute quantification but problems occur in relative quantification as the number of repetitive sequences is unknown. A promising approach was developed where data from amplification of repetitive sequences were used in relative quantification of species in binary mixtures. PCR LUX primers were designed that amplify repetitive and single copy sequences to establish the species dependent number (constants) (SDC) of amplified repetitive sequences per genome. The SDCs and data from amplification of repetitive sequences were tested for their applicability to relatively quantify the amount of chicken DNA in a binary mixture of chicken DNA and pig DNA. However, the designed PCR primers lack the specificity required for regulatory species control.     

泡菜中蛔虫卵荧光PCR检测方法的建立及应用

在模拟阳性泡菜样品中寄生虫卵分离的基础上,本研究通过对单蛔虫卵分别进行加热、液氮/37℃反复冻融、PCR缓冲液冻融、裂解液消化等四种前处理后进行荧光PCR检测,表明将虫卵进行液氮/37℃反复冻融15次后用于荧光PCR检测的效果确实而稳定。所建立的蛔虫卵荧光PCR检测方法特异性好,与鞭虫、毛圆线虫等动物寄生虫线虫不存在交叉反应,可以很好的区分蛔虫与其它动植物线虫卵。敏感性可以满足单个蛔虫卵的检测需要。本方法的建立为泡菜等植物性产品中携带的疑似蛔虫卵的鉴定提供了科学依据。     

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition

Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer’s storage conditions. Sample extraction was performed using a liquid–liquid extraction method prior to LC-MS/MS analysis. AOH was the most prevalent mycotoxins with a 42% at strawberries stored at (22 ± 2)°C and 37% stored at (6 ± 2)°C. The highest AOH levels were found in samples conserved at (22 ± 2)°C ranging between 26 and 752 ng g^–1. AME levels ranged between 11 and 137 ng g^–1, which were found mainly in stored samples at (6 ± 2)°C for more than 28 days. None sample presented levels of TEN in either of the studied conditions.     

Real-time PCR for identification of five species of Cryptolestes based on COI barcode region

Cryptolestes(Coleoptera: Laemophloeidae) are economically important pests, which damage stored products in granaries and mills. Because it is difficult to distinguish Cryptolestes species morphologically, we develop here a high sensitivity real-time PCR method for distinguishing five species of Cryptolestes (C. ferrugineus, C. pusillus, C. turcicus, C. pusilloides and C. capensis). The lower limit of DNA concentration required for species identification is 0.1 ng/μl for C. ferruginues and C. pusilloides, and 0.01 ng/μl for C. pusillus, C. turcicus, and C. capensis. This method successfully determined species assignments for previously unidentified Cryptolestes spp. Collected from the field and will facilitate Cryptolestes identification in the field of plant quarantine and stored product protection.