Pg Of Genomic DNA Research Articles

The development of a species‐specific test to detect Hymenoschyphus pseudoalbidus in ash tissues

SummaryAsh dieback, caused by the pathogen Hymenoscyphus pseudoalbidus, is an emerging lethal disease of Fraxinus excelsior in large parts of Europe. To develop a method for the early detection of H. pseudoalbidus, we designed primers for 46 microsatellites (simple sequence repeats, SSRs) of the pathogen. Seven pairs of primers (SSR38, SSR58, SSR114, SSR198, SSR206, SSR211 and SSR212) were found to bind only to the genome of H. pseudoalbidus, but not to the genome of H. albidus or to 52 different fungal endophytes isolated from F. excelsior and F. angustifolia. Using these seven primer pairs, H. pseudoalbidus was identified in fruiting bodies and different types of ash tissues including dead leaves, dead petioles and discoloured or non‐discoloured wood. Along one twig, H. pseudoalbidus was detected at different levels of intensity, which depended on the distance from symptomatic tissue. The detection limit was 0.9–1.8 pg of genomic DNA per PCR. Of 50 analysed commercially available seedlings, six were infected with H. pseudoalbidus. Two SSR loci (SSR198 and SSR211) showed fragment length polymorphism. Our results showed that the new primers not only provide an easy and inexpensive means of detecting H. pseudoalbidus in ash tissues, but can also provide information on the genetic heterogeneity of the species.

Differential diagnosis of Moniezia benedeni and M. expansa (Anoplocephalidae) by PCR using markers in small ribosomal DNA (18S rDNA)

Moniezia benedeni and M. expansa are common ruminant tapeworms of worldwide distribution, causing gastrointestinal disorders and even death in sheep and goats. In this study, a polymerase chain reaction- (PCR-) based approach for precise species identification was developed and also applied to faecal DNA diagnosis of the tapeworm infection. Since nuclear ribosomal DNA (rDNA) appears to be a useful target for species and/or strain markers, the 18S regions of the rDNA of M. benedeni and M. expansa were amplified and sequenced. The lengths and GC contents of the regions sequenced were 2476-2487 bp and 51.9-52.1% for M. benedeni and 2473 bp and 51.9-52.0% for M. expansa, respectively. Alignment and comparison of the 18S sequences of the two species showed 92.5-93.3% homology. No matches for the 18S regions of M. benedeni and M. expansa were found with other species by BLAST search, which made the 18S sequences appropriate markers for the design of distinctive primers for the two Moniezia species. Our 18S-based PCR could detect as low levels as 0.5 pg genomic DNA or the DNA extracted from 0.2 g faecal sample collected from the rectum of an M. expansa-infected goat. The results indicate that this PCR approach is a reliable alternative for the differential diagnosis of Moniezia species in faecal samples.

AB0781 Clinical periodontal disease predicts the future development of chronic inflammatory arthritis.

BackgroundRecent reports suggest the correlation between rheumatoid arthritis (RA) and periodontal disease (PD). However, it is not clear whether PD affects the disease activity of arthritis or the future introduction...

Fate of the human Y chromosome linked genes and loci in prostate cancer cell lines DU145 and LNCaP

BackgroundProstate cancer is a known cause of mortality in men worldwide although the risk factor varies among different ethnic groups. Loss of the Y chromosome is a common chromosomal abnormality observed in the human prostate cancer.ResultsWe screened 51 standard sequence tagged sites (STSs) corresponding to a male-specific region of the Y chromosome (MSY), sequenced the coding region of the SRY gene and assessed the status of the DYZ1 arrays in the human prostate cancer cell lines DU145 and LNCaP. The MSY was found to be intact and coding region of SRY showed no sequence variation in both the cell lines. However, DYZ1 arrays showed sequence and copy number variations. DU145 and LNCaP cells were found to carry 742 and 1945 copies of the DYZ1, respectively per 3.3 pg of genomic DNA. The DYZ1 copies detected in these cell lines are much below the average of that reported in normal human males. Similarly, the number of “TTCCA” repeat and its derivatives within the DYZ1 arrays showed variation compared to those of the normal males.ConclusionsClearly, the DYZ1 is maximally affected in both the cell lines. Work on additional cell lines and biopsied samples would augment our understanding about the susceptibility of this region. Based on the present work, we construe that copy number status of the DYZ1 may be exploited as a supplementary prognostic tool to monitor the occurrence of prostate cancer using biopsied samples.

Simple and rapid discrimination of embB codon 306 mutations in Mycobacterium tuberculosis clinical isolates by a real-time PCR assay using an LNA-TaqMan probe

Single nucleotide polymorphisms in the codon 306 of embB gene are most frequently reported in ethambutol-resistant Mycobacterium tuberculosis clinical isolates. Here, we report a simple and rapid real-time PCR assay using a locked nucleic acid (LNA)-TaqMan probe for discriminating the embB306 mutations. The use of a 15-bp chimeric LNA/DNA probe led to a relatively higher level of sensitivity and fluorescence signal in the wild-type embB306 ATG codon. Therefore, the mutant alleles were easily distinguishable from the wild-type allele by their distinctive amplification curve shapes without a melting analysis of the PCR product. This system was fast and less than 0.1pg of genomic DNA per reaction was needed for detection. Because the results from this real-time assay were absolutely consistent with those from DNA sequencing, it can be effectively applied as a simple and rapid method for primary screening of embB306 mutations that occur frequently in ethambutol-resistant and/or multidrug-resistant M. tuberculosis isolates.

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real-time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene-producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON- and OTA-producing fungi can be detected and quantified in a single reaction tube using this multiplex real-time PCR method. This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal-based foods.

Development of a specific PCR assay for the rapid and sensitive detection of Phytophthora capsici

Phytophthora blight caused by Phytophthora capsici is a serious disease affecting pepper production and other important vegetable crop. This study developed a species-specific PCR assay for the rapid and accurate detection of P. capsici in infected plant tissues, soil, and water. The PCR primers were designed based on Ras-related protein (Ypt1) gene, and 236 isolates representing 12 species of Phytophthora and 26 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with the species-specific primer pair resulted in products of 364 bp solely from all isolates of P. capsici that was not amplified from any other isolates tested. The detection sensitivity of the species-specific primer pair Pc1F/Pc1R was 10 pg of genomic DNA. This sensitivity increased 1000-fold to 10 fg by developing a nested PCR procedure that uses Pc1F/Pc1R as first-round primers combined with Pc1F/Pc2R as a second round primers. The PCR assay can also be used to detect P. capsici in naturally infected plant tissues, soil, and water. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring and aid in plant disease management.

Evaluating the use of loop-mediated isothermal amplification (LAMP) method for detection of <em>Mycobacterium tuberculosis</em> in Indonesian clinical isolates

Background: Loop-mediated isothermal amplification (LAMP) is a method already claimed as a simple technique to amplify DNA/ RNA using four to six primers as “a set” from conserved sequence of target gene. In this study we optimize the use of LAMP for detection of Mycobacterium tuberculosis in clinical isolates from Indonesia. Methods: Procedures to perform LAMP were optimized, then the method was applied to 122 archieved samples of DNA’s Mtb from clinical TB patients with Acid Fast Bacilli (AFB) smears positive. The samples were obtained in 2008 from 13 provinces in Indonesia for genotyping study, which then become collections of Center for Biomedical and Basic Technology of Health (CBBTH), NIHRD Indonesia. The optimization tests include sensitivity and specificity tests of several sets primers, which were evaluated using 10-fold serially diluted DNA of Mtb H37Rv and 12 species of Mycobacteria. Three equipments consisted of LAMP turbidimeter, heating block and water bath were compared for its ability in DNA amplification. Detection of M. tuberculosis from clinical isolates used set primers specific for gyrB gene, amplicon was detected with UV fluorescence system. Results: The results showed that the highest sensitivity was obtained using the set primers specific for 16S rRNA and gyrB which could detect 10.0 fg to 1.0 pg genomic DNA of Mtb H37Rv. The set primers specific for gyrB gene was the most specific primers. Application of LAMP using gyrB set primers on Indonesian clinical isolates showed 94.2% (114/121) positivity rate. Conclusion: LAMP method is potentially used in TB diagnosis in Indonesia. (Med J Indones. 2012;21:188-95) Keywords: Loop-mediated isothermal amplification, rim gene, 16S rRNA gene, gyrB gene, Mycobacterium tuberculosis

A novel PCR–DNA probe for the detection of fumonisin-producing Fusarium species from major food crops grown in southern India

Fumonisins are a group of 20 chemically related toxic fungal metabolites mainly produced by Fusarium verticillioides and Fusarium proliferatum. In this study, attempt has been made to develop rapid detection of fumonisin-producing Fusarium species from freshly harvested rice and finger millet by novel and sensitive polymerase chain reaction–deoxyribonucleic acid (PCR–DNA) probe. One set of oligonucleotide primers was designed targeting FUM13 gene involved in fumonisin biosynthesis and PCR–DNA probe was developed for dot-blot hybridization assay. The sensitivity of developed PCR–DNA probe was determined; it could detect 10 pg genomic DNA and 1 pg of purified PCR product. To determine the practical usefulness, the developed PCR–DNA probe was evaluated onto the rice grains that were artificially inoculated with toxigenic fungal spores. Sensitivity of the developed probe was found to be 1 × 106 CFU per gram of the rice sample using dot-blot hybridization assay. The specificity of the DNA probe was validated by testing against an array of Fusarium, including Gibberella zeae clade and other fungal strains. All PCR–DNA probe results were further cross-checked with conventional high performance thin layer chromatography (HPTLC) method. PCR–DNA probe results were equivocally matched with the HPTLC method. The novel PCR–DNA probe developed in this study may find application in rapid detection of fumonisin-producing Fusarium isolates from contaminated cereal grains.

Molecular detection of Cylindrocarpon destructans in infected Chinese ginseng roots and soil

Ginseng (Panax ginseng C.A. Meyer) is one of the most important medicinal plants in China, but its yields are often reduced by a variety of root pathogens. The root rot of ginseng is a destructive soilborne disease caused by Cylindrocarpon destructans (teleomorph: Neonectria radicicola). A speciesspecific polymerase chain reaction (PCR) assay was developed for rapid detection of C. destructans in diseased ginseng roots and artificially inoculated soil. One pair of specific primers was designed from comparisons of nucleotide sequences of the nuclear ribosomal internal transcribed spacer (ITS) regions of 22 fungal isolates from northeast of China. Under stringent PCR conditions, primers CD-F and CD-R amplified only a 450 bp fragment from C. destructans DNA but not from other pathogens or negative control. The sensitivity of detection was 1 pg genomic DNA per 25 μl PCR reaction volume, and C. destructans could be specifically detected with CD-F/CD-R from infected ginseng roots and soil. The approach outlined here could be further utilized as a rapid and reliable tool for the diagnosis and monitoring of the root rot of ginseng.

Multiplex PCR assay for the detection of Pantoea stewartii subsp. stewartii using species-specific genetic markers

We developed a multiplex PCR detection assay for quick diagnosis of Pantoea stewartii subsp. stewartii the causal agent of Stewart’s bacterial wilt and leaf blight of maize, using specific genetic markers. The genetic markers of wtsE, cpsA and hrpN gene specifically amplified 519 bp, 375 bp and 200 bp of DNA, respectively, from all isolates of P. stewartii subsp. stewartii but not from other phytopathogenic bacteria. The sensitivity of the genetic markers is down to 103 CFU/mL (5 cells per reaction) of bacterial culture and ~0.5 pg of genomic DNA. In sampling studies, the assay detected P. stewartii subsp. stewartii from artificially infected maize plants 12 days post inoculation. The PCR based genetic markers are specific and they give selective and sensitive amplification of P. stewartii subsp. stewartii for diagnosis in mixed cultures and in planta.

Multiplex quantitative foodborne pathogen detection using high resolution CE‐SSCP coupled stuffer‐free multiplex ligation‐dependent probe amplification

Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen-specific genes are optimal markers. Although multiplex ligation-dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length-dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer-free MLPA using high-resolution CE-SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE-SSCP analysis was designed for species-specific detection. After careful optimization of each MLPA step, to ensure that CE-SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5-5 pg of genomic DNA, and more than 100-fold increase could be quantitatively determined. Thus, MLPA-CE-SSCP is a sensitive and reliable technique for pathogen detection.

Detection of toxigenic Vibrio cholerae with new multiplex PCR

Vibrio cholerae (V. cholerae) is the etiological agent of epidemic cholera. In recent years, cholera has become endemic in different regions of the world. Traditional culture and microscopic methods are considered the standard for the diagnosis of V. cholerae. However, these methods require days for confirmation. Therefore, molecular methods that may be used for the sensitive, accurate, and rapid analysis of V. cholerae are urgently needed. In the present investigation, a multiplex PCR assay was developed for the detection of virulence- and toxigenic-associated (VTA) genes (ctxA, tcpA and ompW). To evaluate PCR specificity, other bacteria from the Enterobacteriaceae family (Salmonella typhi, Shigella dysenteriae, and enterotoxigenic Escherichia coli) and Aeromonas hydrophila were examined. The assay sensitivity was evaluated using colony counting and genome dilution methods. Our results showed that this PCR-based method represents an ideal tool for the rapid detection of VTA genes due to its simplicity, cost effectiveness, and accuracy. This multiplex PCR method can be used to determine the presence of VTA genes and can therefore distinguish V. cholerae bacteria from other Vibrio species and bacteria. This method can detect 10-100 CFU V. cholerae and 8.5-85 pg genomic DNA. This multiplex PCR method has higher sensitivity and specificity than other methods. The proposed test provides an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae.

Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae

In North America, one of the most important root diseases of Poa and Festuca turf is summer patch, caused by Magnaporthe poae. Detection and identification of M. poae in infected roots by conventional culture-based methods is difficult and time consuming, typically taking 3 wk or longer to accomplish. In this study, a culture-independent, TaqMan real-time PCR assay was developed for the detection of M. poae from the roots of fungicidetreated and non-treated Kentucky bluegrass (Poa pratensis) turf. The assay was validated with the target pathogen, closely related fungal species and a number of other microorganisms that inhabit the same host and soil environment. This assay was more sensitive (could detect as little as 3.88 pg genomic DNA of M. poae), rapid and accurate compared to direct microscopic observation and isolation on a selective medium. The real-time PCR detection results corresponded closely to visual assessments of disease severity in the field. Utilization of this assay in diagnostic laboratories will enable turfgrass managers to more quickly and effectively detect and potentially reduce fungicide usage through early and accurate identification of the pathogen.

A Loop-Mediated Isothermal Amplification Method for Rapid Detection of NDM-1 Gene

A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.

Real-Time PCR Assay for Identification of Blastomyces dermatitidis in Culture and in Tissue

The TaqMan real-time PCR assay was developed from the Blastomyces dermatitidis BAD1 gene promoter. The assay identified all haplotypes of B. dermatitidis and five of six positive paraffin-embedded tissues. The assay sensitivity threshold was 1 pg genomic DNA of the mold form and 2 CFU of the yeast form of B. dermatitidis. No cross-reactivity was observed against other fungal DNA. The assay allowed rapid (5-h) identification of B. dermatitidis from culture and from clinical specimens.

Quadruplex real-time polymerase chain reaction assay for molecular diagnosis of Y-chromosomal microdeletions

To develop a rapid and reliable method for molecular diagnosis of Y-chromosomal microdeletions. Study of diagnostic accuracy. Molecular diagnostics laboratories in three hospitals. A total of 701 men with nonobstructive azoospermia or oligozoospermia from three hospitals. We developed a quadruplex real-time polymerase chain reaction (PCR) assay and evaluated its performance in molecular diagnosis of Y-chromosomal microdeletions. Analytic sensitivity, analytic specificity, clinical sensitivity, and clinical specificity. The limit of detection of quadruplex real-time PCR assay was 100 pg genomic DNA. The method attained 100% analytic specificity, 100% clinical sensitivity, and 100% clinical specificity. We have successfully upgraded the diagnostic method published by the European Academy of Andrology and the European Molecular Genetics Quality Network. Our method was validated to be fast, simple, contamination free, of high analytic sensitivity and specificity. Therefore, it is strongly suggested that such quadruplex real-time PCR assay can be readily applied as clinical routine in the near future.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4-6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.

Rapid, specific and quantitative polymerase chain reaction (PCR) detection of pathogenic protozoa Entamoeba histolytica for drinking water supply

In this study, a quantitative species-specific polymerase chain reaction (PCR) method to rapidly detect E. histolytica in water is developed. First, the specificity of E. histolytica PCR detection was verified by using species-specific primers of 16S-like rRNA genes to clearly differentiate it from the closely related amoebae species E. dispar and E. moshkovskii. The sensitivity of this method was subsequently determined using purified E. histolytica genomic DNA and culture cells as PCR reaction templates. Results indicated that conventional PCR visualized on 1% agarose gel was able to detect as low as 0.02 pg genomic DNA and 5 cells, while real-time PCR could detect 0.01 pg genomic DNA and 2 cells of E. histolytica. The protocols for E. histolytica PCR detection in real water samples were then optimized by spiking E. histolytica cells into tap water and reservoir raw water samples. A two-round centrifugation treatment to concentrate amoeba cells directly as a PCR template was the most effective way to detect E. histolytica in spiked tap water samples, while DNA extraction after concentrating amoeba cells was required for spiked reservoir raw water samples. The detection limit of 50 E. histolytica cells in 100 ml tap water was achieved in 2 h from sample collection to real-time PCR data readout. With these established protocols, 78 tap water samples, 11 reservoir raw water samples and 4 feed water samples from Singapore water supply systems were analyzed by both conventional PCR and real-time PCR methods. No E. histolytica cell was detected in tested samples.

Rapid Molecular Assays for Specific Detection and Quantitation of Loa loa Microfilaremia

BackgroundAccurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species.Methodology/Principal FindingsFrom a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50–96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy.Conclusions/SignificanceThis novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection.