scholarly journals Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry

2009 ◽  
Vol 99 (5) ◽  
pp. 582-590 ◽  
Author(s):  
Renaud Ioos ◽  
Céline Fourrier ◽  
Gabriela Iancu ◽  
Thomas R. Gordon
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Fusarium Circinatum ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain ◽  
Seed Dna

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer–probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

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.

scholarly journals Validation of a Real-Time Polymerase Chain Reaction Assay for the Identification of Meloidogyne arenaria

Plant Disease ◽  
2011 ◽  
Vol 95 (7) ◽  
pp. 835-838 ◽  
Author(s):  
Paula Agudelo ◽  
Stephen A. Lewis ◽  
Bruce A. Fortnum
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Soil Samples ◽  
Meloidogyne Arenaria ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain ◽  
Species Specific

Meloidogyne arenaria is an economically important parasite of many crops worldwide. Identification and detection of this species in soil samples is necessary for the design of crop rotation systems, selection of resistant cultivars, and potential use of biological control options. The objective of this study was to develop and validate a real-time polymerase chain reaction (PCR) assay, using species-specific primers and SYBR Green I Dye, for identification of M. arenaria. The specificity of the assay was confirmed by testing for amplification of DNA from other Meloidogyne spp. and from M. arenaria populations of different geographic origins. Field soil samples containing a mixture of M. arenaria and M. incognita were used to compare identification by the real-time PCR assay with identification by esterase phenotype analysis of mature females and by morphometrics of juveniles. The real-time PCR assay provided an accurate and sensitive means for the identification of single juveniles from soil samples.

scholarly journals Identification and Detection of Phoma tracheiphila, Causal Agent of Citrus Mal Secco Disease, by Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2006 ◽  
Vol 90 (12) ◽  
pp. 1523-1530 ◽  
Author(s):  
G. Licciardello ◽  
F. M. Grasso ◽  
P. Bella ◽  
G. Cirvilleri ◽  
V. Grimaldi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Causal Agent ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Phoma Tracheiphila ◽  
Mal Secco

Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal secco causing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore, fast and reliable protocols are required to detect it promptly. A specific primer pair and a dual-labeled fluorogenic probe were used in a real-time polymerase chain reaction (PCR) with the Cepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungus in citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. The sensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimum amount detectable was about 500 fg, whereas the linear quantification range was within 100 ng to 1 pg. Conventional PCR sensitivity was 10 pg. Conventional and real-time PCR successfully detected the fungus in woody samples of naturally infected lemon and artificially inoculated sour orange seedlings. Nevertheless, real-time PCR was about 10- to 20-fold more sensitive than conventional PCR, and preliminary results indicate that the former technique achieves quantitative monitoring of the fungus in tissues. Simple and rapid procedures to obtain suitable DNA samples from fungal cultures and citrus woody samples for PCR assays enable diagnosis to be completed in a short time.

scholarly journals Development of Real-Time Polymerase Chain Reaction Assay with TaqMan Probe for the Quantitative Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus from Shrimp

2006 ◽  
Vol 89 (1) ◽  
pp. 240-244 ◽  
Author(s):  
Zhi-Qin Yue ◽  
Hong Liu ◽  
Wei-Ji Wang ◽  
Zhi-Wen Lei ◽  
Cheng-Zhu Liang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Necrosis Virus ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain

Abstract An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies,with a dynamic range of detection between 9 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-reactwith shrimp genomic DNAor other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

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 Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

2014 ◽  
Vol 34 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Gisele M. Bacanelli ◽  
Carlos A. N. Ramos ◽  
Flábio R. Araújo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Anaplasma Marginale ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
The Real ◽  
End Point ◽  
Polymerase Chain

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

scholarly journals Quantification of Magnaporthe grisea During Infection of Rice Plants Using Real-Time Polymerase Chain Reaction and Northern Blot/Phosphoimaging Analyses

2002 ◽  
Vol 92 (8) ◽  
pp. 870-876 ◽  
Author(s):  
Min Qi ◽  
Yinong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Northern Blot ◽  
Host Resistance ◽  
Real Time Pcr ◽  
Magnaporthe Grisea ◽  
Chain Reaction ◽  
Fungal Pathogenicity ◽  
The Real ◽  
Polymerase Chain

Rice blast, caused by Magnaporthe grisea, is a serious fungal disease of rice worldwide. Currently, evaluation of the fungal pathogenicity and host resistance is mainly based on a disease rating or measurement of blast lesion number and size. However, these methods only provide visual estimation rather than accurate measurement of fungal growth in rice plants. In this study, DNA-based real-time polymerase chain reaction (PCR) and RNA-based northern blot/phosphoimaging analyses were evaluated to quantify M. grisea. Both methods were sensitive, specific, and reproducible and could accurately measure the relative growth and absolute biomass of M. grisea. The real-time PCR analysis showed that the growth of M. grisea in seedling leaves of susceptible cultivars (M201 and Wells) was ≈46 to 80 times higher than that of a resistant cultivar (Drew) at 4 and 6 days after inoculation. The data obtained from the real-time PCR assays also were consistent with that from northern blot/ phosphoimaging analysis. However, the real-time PCR approach was much faster and more convenient in most cases. Therefore, it is an excellent tool for in planta quantification of M. grisea and can be used for reliable assessment of fungal pathogenicity and host resistance

scholarly journals Detection and Quantification of Peronospora tabacina Using a Real-Time Polymerase Chain Reaction Assay

Plant Disease ◽  
2011 ◽  
Vol 95 (6) ◽  
pp. 673-682 ◽  
Author(s):  
Monica Blanco-Meneses ◽  
Jean Beagle Ristaino
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Taqman Assay ◽  
Blue Mold ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Peronospora Tabacina ◽  
The Real ◽  
Tobacco Seedlings ◽  
Polymerase Chain

Peronospora tabacina is an obligate plant pathogen that causes blue mold of tobacco. The disease is difficult to diagnose before the appearance of symptoms and can be easily spread in nonsymptomatic tobacco seedlings. We developed a real-time polymerase chain reaction (PCR) assay for P. tabacina that uses 5′ fluorogenic exonuclease (TaqMan) chemistry to detect and quantify pathogen DNA from diseased tissue. The primers and probe were designed using 5.8S ribosomal DNA sequences from 12 fungal and oomycete tobacco pathogens and 24 Peronospora spp. The PtabBM TaqMan assay was optimized and performed with a final concentration of 450 nM primers and 125 nM probe. The real-time TaqMan assay was assessed for sensitivity and the lower detection limit was 1 fg of DNA. The assay was specific for P. tabacina. None of the DNA from other tobacco pathogens, nonpathogens, or the host were amplified. The PtabBM TaqMan assay was useful for detection of P. tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings. The assay was used to quantify host resistance and it was possible to detect the pathogen 4 days postinoculation in both medium-resistant and susceptible tobacco cultivars. The real-time PCR assay for P. tabacina will be a valuable tool for the detection of the pathogen and of use to regulatory agencies interested in preventing the spread of blue mold.

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.

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