scholarly journals Simultaneous Detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization and Real-Time Polymerase Chain Reaction

2009 ◽  
Vol 99 (6) ◽  
pp. 666-678 ◽  
Author(s):  
Y. Ha ◽  
A. Fessehaie ◽  
K. S. Ling ◽  
W. P. Wechter ◽  
A. P. Keinath ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Threshold ◽  
Simultaneous Detection ◽  
Didymella Bryoniae ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Magnetic Capture ◽  
Polymerase Chain ◽  
Pcr Assays

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 105 conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/μl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

Early and simultaneous detection ofNosema bombycis(Microsporidia: Nosematidae), nucleopolyhedrovirus (Baculoviridae), and densovirus (Parvoviridae) by multiplex real-time polymerase chain reaction inBombyx mori(Lepidoptera: Bombycidae)

2017 ◽  
Vol 149 (2) ◽  
pp. 265-275
Author(s):  
Shan Wu ◽  
Yong-Qiang He ◽  
Xing-Meng Lu ◽  
Xiao-Feng Zhang ◽  
Jiang-Bing Shuai ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Early Detection ◽  
Bombyx Mori ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Post Inoculation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

AbstractAn effective multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of three major pathogens,Nosema bombycisNägeli (Microsporidia: Nosematidae),Bombyx morinucleopolyhedrovirus (Baculoviridae: genusAlphabaculovirus) (NPV), andBombyx moridensovirus (Parvoviridae: genusIteravirus) (DNV), in silkworms (Bombyx mori(Linnaeus); Lepidoptera: Bombycidae) was developed in this study. Polymerase chain reaction and real-time PCR tests and basic local alignment search tool searches revealed that the primers and probes used in this study had high specificities for their target species. The ability of each primer/probe set to detect pure pathogen DNA was determined using a plasmid dilution panel, in which under optimal conditions the multiplex real-time PCR assay showed high efficiency in the detection of three mixed target plasmids with a detection limit of 8.5×103copies forN. bombycisandBombyx moriNPV (BmNPV) and 8.5×104copies forBombyx moriDNV (BmDNV). When the ability to detect these three pathogens was examined in artificially inoculated silkworms, our method presented a number of advantages over traditional microscopy, including specificity, sensitivity, and high-throughput capabilities. Under the optimal volume ratio for the three primer/probe sets (3:2:2=N. bombycis:BmNPV:BmDNV), the multiplex real-time PCR assay showed early detection of BmNPV and BmDNV by day 1 post inoculation using DNA templates of the three pathogens in various combinations from individually infected silkworms; the early detection ofN. bombyciswas possible by day 3 post inoculation using the DNA isolated from the midgut ofN. bombycis-infected silkworms.

scholarly journals Early Detection of Leifsonia xyli subsp. xyli in Sugarcane Leaves by Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 430-434 ◽  
Author(s):  
M. P. Grisham ◽  
Y.-B. Pan ◽  
E. P. Richard
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Close Agreement ◽  
Leaf Tissue ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Pcr Assays

A real-time, polymerase chain reaction (PCR) assay was developed for detecting Leifsonia xyli subsp. xyli in sugarcane leaf tissue. Real-time PCR assays were conducted on the youngest, fully expanded leaf of three cultivars collected bi-weekly from field nurseries between 11 April and 19 July 2005. L. xyli subsp. xyli infection was detected in leaves collected at all sampling dates, including those from 1-month-old plants on 11 April. Assays conducted on older, more rapidly growing plants (28 July and 21 October 2005) indicated that leaf position affects assay efficiency. Conventional PCR was less efficient than real-time PCR for detecting L. xyli subsp. xyli in leaf tissue. Real-time PCR was used to rank cultivars for susceptibility to L. xyli subsp. xyli infection based on the relative titer of L. xyli subsp. xyli in leaves of inoculated, 3- and 4-month-old greenhouse-grown plants. The ranking of cultivars by real-time PCR was in close agreement with the ranking determined by tissue-blot enzyme immunoassay performed on tissue from 7- to 9-month-old stalks.

A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

2021 ◽  
Author(s):  
Yang Pan ◽  
Jing Chen ◽  
Junhuang Wu ◽  
Yongxia Wang ◽  
Junwei Zou ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rapid Detection ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Canine Kobuvirus

Abstract Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

Detection and Quantitation of Enterohemorrhagic Escherichia coli O157, O111, and O26 in Beef and Bovine Feces by Real-Time Polymerase Chain Reaction†

2002 ◽  
Vol 65 (9) ◽  
pp. 1371-1380 ◽  
Author(s):  
VIJAY K. SHARMA
Keyword(s):  
Real Time ◽  
Shiga Toxin ◽  
Enterohemorrhagic Escherichia Coli ◽  
Specific Primer ◽  
Chain Reaction ◽  
Pcr Assay ◽  
E Coli ◽  
Taqman Probes ◽  
Polymerase Chain ◽  
Pcr Assays

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and certain non-O157 EHEC serotypes (such as O26:H11, O26: NM, O111:H8, and O111:NM) have emerged as significant causes of human disease throughout the world. Important virulence attributes of EHEC are the intimin protein (encoded by the eae gene) and Shiga toxins 1 and 2 (encoded by the stx1 and stx2 genes, respectively). Two sets of real-time polymerase chain reaction (R-PCR) assays were developed for the simultaneous detection and quantitation of EHEC through the monitoring of the presence of the eae and stx genes, and these assays were evaluated. In the eaeR-PCR assay, three sets of primers and TaqMan probes were designed for the amplification and real-time detection of a portion of the eae gene specific to the EHEC O26, O111, and O157 serotypes. In the stxR-PCR assay, two sets of primers and TaqMan probes were used to amplify and detect the stx1 and stx2 genes. DNA prepared from 67 bacterial strains carrying known virulence markers was tested to determine the specificities of the two assays. In the eaeR-PCR assay, eaeO157- and eaeO111-specific primer-probe sets identified only EHEC O157 and O111 strains, respectively. The eaeO26-specific primer-probe set identified all EHEC O26 isolates and some Shiga toxin–negative serotypes of enteropathogenic E. coli and rabbit diarrheagenic E. coli. The stxR-PCR assay was able to identify only those strains carrying either or both of the Shiga toxin–encoding genes. The detection range of both R-PCR assays was linear over DNA concentrations corresponding to 103 to 108 CFU/ml of an EHEC strain. Both assays were able to detect and quantify very low levels (1 to 10 CFU/g of food or feces) of EHEC in feces and ground beef enriched for 16 h in a modified Trypticase soy broth. In conclusion, eae- and stx-based R-PCR assays are reliable and sensitive methods for the rapid screening and specific and quantitative detection of important serotypes of EHEC in cattle and in foods of bovine origin.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

scholarly journals Influenza species and subtypes circulation among hospitalized patients in Laleh hospital during two influenza seasonal (2016-2017 and 2017-2018) using a multiplex Real Time-Polymerase Chain Reaction

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Iman Rezaee Azhar ◽  
Minoo Mohraz ◽  
Masoud Mardani ◽  
Mohammad Ali Tavakoli ◽  
Amin Ehteshami Afshar ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Hospitalized Patients ◽  
P Value ◽  
Chain Reaction ◽  
Short Period ◽  
The Mean ◽  
Polymerase Chain

The introduction of polymerase chain reaction (PCR) techniques has improved the detection of respiratory viruses, particularly with the use of multiplex real-time technique with the capability of simultaneous detection of various pathogens in a single reaction. The aim of this study was to apply the above technology for the diagnosis of influenza infections and at the same time to differentiate between common flu species between hospitalized patients in Laleh hospital (Iran) between two flu seasons (2016- 2017 and 2017-2018). Different respiratory specimens were collected from 540 patients from a period of December 2016 to May 2018 and were sent to the laboratory for molecular diagnosis. RNAs were extracted and subsequently, a multiplex real time PCR identifying flu A, flu B and typing flu A (H1N1) was carried out. The mean age of patients was 47.54±23.96. 216 (40%) and 321 (60%) of subjects were male and female, respectively. 219 out of 540 (40.5%) were positive for influenza infection including flu A (n=97, 44.3%), flu A (H1N1) (n=45, 20.7%) and flu B (n=77, 35%). Flu A was the dominant species on 2016-2017 and flu B was the major species on 2017-2018. Flu A (H1N1) was comparable in both time periods. Flu infections were most frequently diagnosed in age groups 21-40. Flu-positive patients suffered more from body pain and sore throat than flunegative patients with significant statistical difference (P values <0.001). The mean duration of hospitalization was shorter for flu-positive patients (P value = 0.016). Application of multiplex real time PCR could facilitate the influenza diagnosis in a short period of time, benefiting patients from exclusion of bacterial infections and avoiding unnecessary antibiotic therapy. Influenza diagnosis was not achieved in up to 60% of flu-like respiratory infections, suggesting the potential benefit of adopting the same methodology for assessing the involvement of other viral or/and bacterial pathogens in those patients.

A multiplex real-time PCR assay for the simultaneous quantification of the major plant-parasitic nematodes in Japan

Nematology ◽  
2011 ◽  
Vol 13 (6) ◽  
pp. 713-720 ◽  
Author(s):  
Yu Yu Min ◽  
Keita Goto ◽  
Koki Toyota ◽  
Erika Sato
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Single Species ◽  
The Other ◽  
Parasitic Nematodes ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Pcr Assays

AbstractMultiplex real-time PCR assays were developed to quantify multiple species of Meloidogyne incognita, Pratylenchus penetrans, Globodera rostochiensis and Heterodera glycines in soil. The probes specific for P. penetrans and H. glycines are labelled with a fluorescence molecule, FAM, and those for M. incognita and G. rostochiensis with ROX. The primers and probes are species-specific to P. penetrans, but group-specific to the other species. DNA was extracted from suspensions containing each nematode and multiplex Cycleave® PCR assays were done for pairs of P. penetrans and M. incognita, P. penetrans and G. rostochiensis, or G. rostochiensis and H. glycines. The results revealed that the target nematode, except for H. glycines, was quantified in the presence of less than 100 times that of the other nematode (competitor), but underestimated in the presence of 1000 times the competitor. Such underestimation was solved by the use of SYBR Green I real time PCR assays targeting a single species. Multiplex PCR assay for P. penetrans and M. incognita was done using environmental DNA (eDNA) extracted from a soil naturally infested with the nematodes. Results quantified both species. Multiplex assay using eDNA may enable a sensitive and simultaneous detection of P. penetrans and M. incognita or P. penetrans and G. rostochiensis in soil although caution is needed in case the existing ratio is biased to one of the species.

scholarly journals A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

2006 ◽  
Vol 96 (11) ◽  
pp. 1255-1262 ◽  
Author(s):  
C. Zijlstra ◽  
R. A. Van Hoof
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Simultaneous Detection ◽  
Chain Reaction ◽  
Meloidogyne Chitwoodi ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.

SLCO1B1 c.521T>C Genotyping in the Austrian Population Using 2 Commercial Real-Time Polymerase Chain Reaction Assays: An Implementation Study

Pharmacology ◽  
2018 ◽  
Vol 102 (1-2) ◽  
pp. 88-90
Author(s):  
Dietmar Enko ◽  
Sophia Harringer ◽  
Christian Oberkanins ◽  
Helene Pühringer ◽  
Gabriele Halwachs-Baumann ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Performance ◽  
Chain Reaction ◽  
Increased Risk ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Austrian Population ◽  
Genotype And Allele Frequencies

Statin-induced myopathy is reported to be significantly associated with the SCLO1B1 c.521T>C polymorphism. To date, SLCO1B1 c.521T&gt;C epidemiologic data for the Austrian population is still lacking. Therefore, this study aimed at assessing the genotype and allele frequencies of the SLCO1B1 c.521T&gt;C variant in Austria and evaluating the clinical performance of 2 commercial real-time polymerase chain reaction (PCR) assays. Genomic DNA isolated from 181 healthy individuals was analyzed for the SLCO1B1 c.521T&gt;C polymorphism in a comparative manner using the SLCO1B1 c.521T&gt;C RealFastTM Assay and the BioPro SLCO1B1 Genotyping real-time PCR Kit. A total of 10 (5.5%) and 44 (24.3%) out of 181 individuals were SLCO1B1 c.521T&gt;C C/C-homo- and ­C/T-heterozygotes, the genotypes indicative of high and increased risk of statin-induced myopathy, respectively. The SLCO1B1 c.521C allele frequency rate was 17.7%. In conclusion, the genetic predisposition of elevated statin-induced myopathy risk in the Austrian population is frequent. Both real-time PCR assays under investigation here are reliable and robust SLCO1B1 c.521T&gt;C genotyping tools in clinical routine.

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