Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR

AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR. CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.     

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.     

Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/ HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.     

Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5' nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be >/=10(2) CFU/ml in pure cultures and >/=10(3) CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5' nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.     

Characterization of newly developed SSLP markers for the rat

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27–36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F₂ progeny of (OLETFxBN)F₂ crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species. Received: 8 July 1999 / Accepted: 3 December 1999     

文蛤微卫星DNA的筛选及其特性分析

采用磁珠富集分离法从文蛤(Meretrix meretrix)的基因组中筛选得到49条微卫星DNA序列,其中两碱基重复有3种类型36个序列,四碱基重复有14种类型26个序列.重复次数在5～30之间的序列占75.6%,30次以上重复的序列占24.4%,最高重复次数为100.根据重复单元的排列特点,完美型、非完美型及混合型序列所占比例分别为61.2%、14.5%和24.5%.本研究中构建的文蛤微卫星文库将在文蛤种质资源评价及分子遗传学研究中发挥作用.     

Prevalence of pathogenic Yersinia enterocolitica strains in pigs in the United States

Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United States using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5' nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P < 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans.     

Factors affecting the performance of 5' nuclease PCR assays for Listeria monocytogenes detection

The design and operating parameters affecting the performance of 5' nuclease PCR (TaqMan) assays for the detection of Listeria monocytogenes was investigated. A system previously developed and based on the hlyA gene was used as a model [Appl. Environ. Microbiol. 61 (1995) 3724]. A series of fluorogenic probes labeled with a reporter and a quencher dye was synthesized to explore the effect of probe position and sequence content on the efficiency of probe hydrolysis. In addition, a series of PCR primer pairs that altered the distance between the upstream primer and the interceding probe was examined. The effects of various assay parameters were evaluated by measuring the ratio of the fluorescence intensity of the reporter dye over the quencher dye (deltaRQ). For a given probe sequence, the deltaRQ was typically lower if the 5' terminus was a G residue. Decreasing the probe concentration increased the deltaRQ, although this was at the expense of reproducibility in the assay readout. The distance between the upstream primer and the interceding probe has a significant effect on probe hydrolysis. Reducing the primer-probe distance from, for example, 127 to 4 nt increased the deltaRQ from 2.87 to 5.00. These general rules were used to develop a 5' nuclease PCR (TaqMan) assay with enhanced signal output, providing higher and more reproducible deltaRQ values for L. monocytogenes detection.     

Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

Allelic discrimination using fluorogenic probes and the 5′ nuclease assay

Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5′ nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5′ nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.     

Rapid detection of CA polymorphisms in cloned DNA: application to the 5'' region of the dystrophin gene.

To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.     

毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR

A real-time PCR method using a fluorogenic 5' nuclease assay and a PE Applied Biosystems GeneAmp 5700 sequence detector was developed to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. A pair of PCR primers to amplify an 81 bp DNA fragment and a fluorogenic probe (TaqMan probe) were selected from ORF1 (open reading frame 1) of the IHHNV genome. The primers and TaqMan probe used in this assay were shown to be specific for IHHNV and did not react with either hepatopancreatic parvovirus (HPV), white-spot syndrome virus (WSSV), or shrimp DNA. A plasmid, pIHHNV-P4, containing the target IHHNV sequence was constructed and used as a positive control. The concentration of pIHHNV-P4 was determined through spectrophotometric analysis and the plasmid was used for quantitative studies. This real-time PCR assay had a detection limit of 10 copies and a log-linear range up to 5 x 10(7) copies of IHHNV DNA. The assay was then used to quantify IHHNV in infected shrimp collected from 5 locations: Hawaii, Panama, Mexico, Guam, and the Philippines. The quantitative analysis showed that wild-caught, large juvenile Penaeus stylirostris collected from the Gulf of California (Mexico) in 1996 were naturally infected with IHHNV and contained up to 10(9) copies of IHHNV microg(-1) of DNA. Similar quantities of IHHNV were detected in hatchery-raised, small juvenile P. stylirostris collected from Guam in 1995 and in farm-raised, post-larval P. monodon from the Philippines in 1996. Laboratory-infected P. stylirostris contained approximately 10(8) copies of IHHNV 31 d after being fed with IHHNV-infected shrimp tissue. In contrast, individuals of Super Shrimp, a line of P. stylirostris selected for IHHNV resistance, showed no signs of infection 32 d after ingesting IHHNV-infected shrimp tissue. Laboratory-infected P. vannamei also contained approximately 10(8) copies of IHHNV 30 d after being fed infected shrimp tissue. A time-course study of IHHNV replication in juvenile P. vannamei showed that the doubling time in the exponential growth phase was approximately 22 h.     

miR-451a和miR-21-5p双重实时荧光定量PCR检测体系的建立及应用

目的 为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR (real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法 设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果 优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论 该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。     

An integrated comparative map of the porcine X chromosome

The objectives of this study were to assign both microsatellite and gene-based markers on porcine chromosome X to two radiation hybrid (RH) panels and to develop a more extensive integrated map of SSC-X. Thirty-five microsatellite and 20 gene-based markers were assigned to T43RH, and 16 previously unreported microsatellite and 15 gene-based markers were added to IMpRH map. Of these, 30 microsatellite and 12 gene-based markers were common to both RH maps. Twenty-two gene-based markers were submitted to BLASTN analysis for identification of orthologues of genes on HSA-X. Single nucleotide polymorphisms (SNPs) were detected for 12 gene-based markers, and nine of these were placed on the genetic map. A total of 92 known loci are present on at least one porcine chromosome X map. Thirty-seven loci are present on all three maps; 31 loci are found on only one map. Location of 33 gene-based markers on the comprehensive map translates into an integrated comparative map that supports conservation of gene order between SSC-X and HSA-X. This integrated map will be valuable for selection of candidate genes for porcine quantitative trait loci (QTLs) that map to SSC-X.     

Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5′ nuclease assay

The presence of Listeria monocytogenes as a dairy food contaminant is a lethal threat to dairy industrialists; therefore, products tainted with L. monocytogenes must be quickly detected and removed from production. This fluorogenic PCR-based assay was developed to rapidly detect L. monocytogenes contamination in dairy samples before a final product is distributed. The detection method employed uses a PCR primer pair and a fluorogenic TaqMan probe which bind to a region of a virulence determinant gene specific to L. monocytogenes. As the DNA target is amplified, the 5′ nuclease activity of Taq DNA polymerase hydrolyzes the internal fluorogenic probe creating a change in fluorescence that can be monitored and automatically analyzed with a fluorometer. Sensitivity studies indicated a lower detection limit of under 10 CFU for pure culture extracts and spiked dairy enrichments. A study was performed on 266 dairy product samples obtained from Central California dairy production plants. Eighty-three of these samples were artificially spiked with both high and low concentrations of L. monocytogenes before an overnight enrichment in TSB/LiCl/colostin sulfate/moxalactam media. DNA from enriched samples was obtained using a rapid Chelex extraction specifically designed for dairy sample enrichments and automated analysis. The extraction was followed by the fluorogenic PCR assay and measurement of fluorescence increase. The assay was completed within 24 h, with an observed 95.2% sensitivity, 96.7% specificity, 92.9% positive predictive value, 97.8% negative predictive value, and 96.2% accuracy. According to specificity studies, five other bacterial species cross-reacted with the fluorogenic 5′ nuclease PCR. However, only one of these strains (Listeria grayi) was able to grow in the enrichment medium employed, and was not isolated from any of the 266 dairy product enrichments evaluated in this study. Therefore, this method provides a rapid, sensitive, and automatable analysis alternative to standard culture techniques for the detection of Listeria monocytogenes in dairy samples. Received 4 February 1998/ Accepted in revised form 1 October 1998     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

Assessment of AFLP marker behaviour in enriching STS radiation hybrid maps

Radiation hybrid (RH) mapping provides a powerful tool to build high-resolution maps of genomes. Here, we demonstrate the use of the AFLP^® technique for high-throughput typing of RH cell lines. Cattle were used as the model species because an RH panel was available to investigate the behaviour of AFLP markers within the microsatellite- and STS-based maps of this species. A total of 747 AFLP markers were typed on the TM112 RH radiation panel and 651 of these were assigned by two-point analysis to the 29 bovine autosomes and sex chromosomes. AFLP markers were added to the 1222 microsatellite and STS markers that were included in earlier RH maps. Multipoint maps were constructed for seven example chromosomes, which retained 248 microsatellite and STS markers, and added 123 AFLP markers at LOD 4. The addition of the AFLP markers increased the number of markers by 42.1% and the map length by 10.4%. The AFLP markers showed lower retention frequency (RF) values than the STS markers. The comparison of RF values in AFLP markers and their corresponding AFLP-derived STSs demonstrated that the lower RF values were due to the lower detection sensitivity of the AFLP technique. Despite these differences, AFLP and AFLP-derived STS markers mapped to identical or similar positions. These results demonstrate that it is possible to merge AFLP and microsatellite markers in the same map. The application of AFLP technology could permit the rapid construction of RH maps in species for which extensive genome information and large numbers of SNP and microsatellite markers are not available.