Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the β-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.     

不同类型油菜EST-SSR标记的通用性及其应用研究

以10个甘蓝型油菜、11个芥菜型油菜和7个白菜型油菜品种为材料,选用已报道的14对白菜EST-SSR引物和8对油菜EST-SSR引物,探索其在3种类型油菜中的通用性,并利用筛选出的在3类油菜中有通用性和多态性的EST-SSR标记对供试油菜品种进行聚类分析.结果表明:(1)选用的14对白菜EST-SSR引物和8对油菜EST-SSR引物,在供试的3种类型油菜中都有扩增,完全可用.(2)在白菜型、甘蓝型和芥菜型油菜品种中扩增显示出多态性的引物数分别为18、16 和14对,其中有10对引物在3种类型油菜品种间扩增产物具有多态性.(3)利用这10对扩增多态性EST-SSR引物对供试油菜品种聚类分析,结果显示,在遗传相似系数为0.67时,3种类型油菜品种分别独自聚为一大类,表明开发和建立不同类型油菜间可通用的EST-SSR标记是可行且有应用价值的.     

油菜EST-SSR标记的建立

在油菜17987条非冗余EST中,共发掘出了2083个EST-SSR,分布于2443条EST中,发生频率是13.58%,平均分布距离为4.34kb。在油菜EST-SSR中,二、三核苷酸重复是主要的重复类型,二者出现的频率相近,占总SSR的89.05%。AG/CT和AAG/CTT是二、三核苷酸中的优势重复类型,分别占二、三核苷酸重复类型的84.31%和37.71%。进一步设计了23对SSR引物,通过梯度PCR试验确定了各引物的适宜退火温度,并利用非变性聚丙烯酰胺凝胶银染对这些引物在10个油菜品种中的扩增情况和多态性进行了检测。有21对引物显示扩增,引物可用率为91.30%;有12对引物显现出多态性,占可扩增引物的57.14%。本研究结果证明根据油菜EST建立SSR标记是有效、可行的。     

Early detection of white mold caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in potato fields using real-time PCR

Potato white mold caused by Sclerotinia sclerotiorum is an important plant disease occurring in many potato-producing areas worldwide. This disease causes considerable yield losses in a large number of potato fields of Hamedan province, Iran. Although several non-chemical methods have been suggested against the pathogen, application of chemicals is considered a major method employed by the growers in Hamedan fields leading to considerable pollution of underground water. Therefore, it is necessary to find techniques to help growers make a more timely decision for chemical application. In this research, a pair of PCR primers was designed according to the nucleotide sequence of a region of DNA amplified by the primer M13. This primer set amplified a 352-bp fragment from all isolates of S. sclerotiorum collected from Hamedan, Iran, and Hangzhou, China. However, this situation was not observed in other tested fungi. A real-time PCR assay was conducted to detect early infection of the pathogen on potato petals. This real-time PCR assay is suggested for growers to make timely decisions on chemical application.     

Quantification of host-specific <Emphasis Type="Italic">Bacteroides–Prevotella</Emphasis> 16S rRNA genetic markers for assessment of fecal pollution in freshwater

Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides–Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9–11 log₁₀ copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides–Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r ² = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50–800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.     

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

油菜内生细菌16S核糖体DNA的RFLP分析

植物内生细菌定殖在植物组织内部但不引起明显的病害症状。从健康油菜植株的不同器官中分离到大量内生细菌,这些细菌菌落形态存在明显的差异,表明油菜组织中存在大量内生细菌,且类群丰富。分离到的122株内生细菌根据菌落形态可以划分为35类;利用细菌通用引物对16S核糖体DNA进行扩增, 获得约15 kb片段,分别用内切酶HaeⅢ和MspⅠ对扩增产物进行限制性酶切,产生不同的酶切图谱,根据酶切图谱聚类分析结果,所有供试菌株被归为39类,这一结果从遗传上显示油菜内生细菌类群的多样性。两种方法归类结果比较发现菌落特征所反映的信息量有限,只能作为初步的参考指标,核糖体DNA的限制性片段长度多态性分析快速、准确,可以作为油菜内生细菌多样性分析的一种有效方法     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.     

In vivo measurements of changes in pH triggered by oxalic acid in leaf tissue of transgenic oilseed rape

Oxalic acid (OA), a non-host-specific toxin secreted by Sclerotinia sclerotiorum during pathogenesis, has been demonstrated to be a major phytotoxic and pathogenic factor. Oxalate oxidase (OXO) is an enzyme associated with the detoxification of OA, and hence the introduction of an OXO gene into oilseed rape (Brassica napus L.) to break down OA may be an alternative way of increasing the resistance of the plant to Sclerotinia sclerotiorum. In order to investigate the activation of OXO in transgenic oilseed rape, a convenient and accessible method was used to monitor changes in pH in response to stress induced by OA. The pH sensor, a platinum microcylinder electrode modified using polyaniline film, exhibited a linear response within the pH range from 3 to 7, with a Nernst response slope of 70 mV/pH at room temperature. The linear correlation coefficient was 0.9979. Changes induced by OA in the pH values of leaf tissue of different oilseed rape species from Brassica napus L. were monitored in real time in vivo using this electrode. The results clearly showed that the transgenic oilseed rape was more resistant to OA than non-transgenic oilseed rape.     

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.     

基因结构分析软件对实时定量PCR引物进行设计和筛选

目的：以人丝裂原活化蛋白激酶3(mitogen-activated protein kinase 3, ) 基因结构为例,利用不同生物相关软件分析、 设计和筛选合适的定量PCR 引物。方法：利用NCBI 的Gene 数据库查找人基因的参考序列、UniGene 数据库查找标准 参考序列;并用在线软件如Spidey, UCSC, Ensembl 等分析基因结构;利用Primer3,Oligo6,IDT 等软件进行引物设计;用MFOLD 程序分析基因二级结构后,选择引物可定位的外显子位置;利用电子PCR进行引物扩增特异性的检验;最后通过实验检验引物的 扩增效果。结果：从程序软件推荐的引物列表中筛选出一对能特异扩增人基因的引物。结论：基因结构分析软件有助于定 量PCR 引物的设计。     

Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.     

Real-time PCR Assay Based on Topoisomerase-II Gene for Detection of <Emphasis Type="Italic">Fusarium udum</Emphasis>

Fusarium wilt is an important soilborne disease of pigeonpea, caused by Fusarium udum. In this study, we have designed a real-time PCR assay for the detection of Fusarium udum from infected pigeonpea plants. Based on Topoisomerase-II gene sequence data from Fusarium udum and other related Fusarium species, a pair of primer was designed. The species-specific primers were tested in real-time PCR SYBR green assay. No increasing fluorescence signals exceeding the baseline threshold was observed with tested microbes, except Fusarium udum DNA. A single dissociation peak of increased fluorescence was obtained for the specific primers at melting temperature of 81.25°C. The real-time PCR showed a lowest detection of 0.1 pg genomic DNA. The assay was more sensitive, accurate and less time consuming for detection of Fusarium udum in infected plants root.     

Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood

Brucellosis is a widespread zoonosis. Currently the diagnosis of this zoonosis is based on microbiological and serological laboratory tests. Polymerase chain reaction (PCR) has been used to detect DNA from Brucella. Different target genes, primer pairs, PCR techniques, and extraction procedures have previously been published for Brucella detection. But only a few of these primers have been used in human samples, and only one study has been carried out to compare sensitivity between them. In the present study, 3 sets of primers and 3 different PCR protocols amplifying 3 different regions of the Brucella genome were compared for detection of Brucella DNA in a peripheral-blood PCR assay to conclude which is most suitable for the clinical diagnostic laboratory. These 3 pairs of primers amplify 3 different fragments included in (i) a gene encoding a 31 kDa Brucella abortus antigen (B4/B5), (ii) a sequence 16S rRNA of B. abortus (F4/R2), and (iii) a gene encoding an outer membrane protein (omp-2) (JPF/JPR). Some modifications on the reported techniques were applied during the present work to improve the outcome. The results showed that the B4/B5 primer pair had the highest sensitivity for detection of positive samples (98%), the JPF/JPR primer pair detected 88.4% of positive samples, whereas F4/R2 primer pair was the least sensitive, being able to detect only 53.1% of positive samples. The specificity of the 3 techniques was 100%. The B4/B5 primer pair was also able to detect the smallest number of bacteria (700 cfu/mL), whereas JPF/JPR was able to detect 7 x 105 cfu/mL and F4/R2 was able to detect 7 x 107 cfu/mL. It is thus concluded that using the B4/B5 primer PCR with the suggested modifications is a robust assay, which meets the sensitivity requirements to be used for testing of human blood samples for brucellosis in the diagnostic laboratory.     

Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains

For the detection of pathogenic Yersinia enterocolitica strains, a duplex PCR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtained by using a primer derived from the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences. From the sequence of one pathogen-specific amplified fragment, a discriminative primer has been designed bridging the sequence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected open reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocolitica and to discriminate between pathogenic and non-pathogenic strains. The same primer combinations were used in an on line rapid cycling real-time PCR assay. The used SYBR Green I format allowed for the easy translation of the PCR conditions and confirmation of the resulting amplicons. The time of analysis was reduced to approximately 60 min.