scholarly journals Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Sheng-Ren Sun ◽  
Kashif Ahmad ◽  
Xiao-Bin Wu ◽  
Jian-Sheng Chen ◽  
Hua-Ying Fu ◽  
...  
Keyword(s):  
Real Time ◽  
Leaf Tissue ◽  
High Specificity ◽  
Rnase H ◽  
Conventional Pcr ◽  
Tissue Samples ◽  
Real Time Quantitative Pcr ◽  
Yield And Quality ◽  
Sugarcane Germplasm ◽  
First Time

Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs.

scholarly journals Adapting a conventional pcr assay forToxoplasma gondiidetection to real-time quantitative pcr including a competitive internal control

Parasite ◽  
2007 ◽  
Vol 14 (2) ◽  
pp. 149-154 ◽  
Author(s):  
M.P. Brenier-Pinchart ◽  
V. Morand-Bui ◽  
H. Fricker-Hidalgo ◽  
V. Equy ◽  
R. Marlu ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Internal Control ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr

scholarly journals Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine

2006 ◽  
Vol 72 (11) ◽  
pp. 7148-7155 ◽  
Author(s):  
Núria Hierro ◽  
Braulio Esteve-Zarzoso ◽  
Ángel González ◽  
Albert Mas ◽  
Jose M. Guillamón
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Acetic Acid Bacteria ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr ◽  
Yeast Extract Peptone Dextrose ◽  
Different Strains ◽  
26S Rrna ◽  
First Time

ABSTRACT Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

scholarly journals Design and validation of oligonucleotide primers suitable for waterborne bacterial pathogen detection via real-time qPCR

2021 ◽  
Author(s):  
Shawn Thomas Clark
Keyword(s):  
Real Time ◽  
Pathogen Detection ◽  
Extraction Procedure ◽  
Microbiological Quality ◽  
Bacterial Pathogen ◽  
High Specificity ◽  
Fecal Coliforms ◽  
Oligonucleotide Primers ◽  
Real Time Quantitative Pcr ◽  
Quality Of Water

Fecal coliforms have been used as indicators to evaluate health risks associated with the microbiological quality of water for many years. Recent studies have challenged their ability to accurately predict bacterial numbers in the natural environment. DNA-based assays are proposed candidates to replace existing methods, but protocols suited for standardized direct-use have not yet been sufficiently developed. The objective of this study was to examine the feasibility of using real-time quantitative PCR (qPCR) to detect contamination from five waterborne bacterial pathogens in surface and treated drinking waters. Robust oligonucleotide primers were assembled to target virulence-associated genes. Primers were found to have high specificity and increased sensitivity for low pathogen loads of 10 cells/mL, as determined experimentally via qPCR. Detection of pathogenic cells directly from an environmental matrix has also been demonstrated using a filtration-extraction procedure. The developed protocols have shown their potential for use in conjunction with traditional indicator techniques.

scholarly journals Molecular Epidemiology of Ovine Papillomavirus Infections Among Sheep in Southern Italy

2021 ◽  
Vol 8 ◽  
Author(s):  
Francesca De Falco ◽  
Anna Cutarelli ◽  
Nicola D'Alessio ◽  
Pellegrino Cerino ◽  
Cornel Catoi ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Real Time ◽  
Molecular Diagnostic ◽  
Multiple Infections ◽  
Blood Samples ◽  
Real Time Quantitative Pcr ◽  
Healthy Sheep ◽  
Digital Polymerase Chain Reaction ◽  
First Time ◽  
Real Time Qpcr

Ovine papillomaviruses (OaPVs) were detected and quantified, for the first time, using droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (qPCR) via blood samples of 165 clinically healthy sheep. OaPV DNA was detected in 126 blood samples (~76.4%). DdPCR detected OaPV DNA in 124 samples; in only two additional samples positive for real-time qPCR, ddPCR failed to detect the presence of any OaPVs. In 70 of the positive samples (~55.6%), a single OaPV infection was observed, 12 of which were caused by OaPV1 (~17.1%) and 14 by OaPV2 (20%). OaPV3 was responsible for 19 single infections (~27.1%), and OaPV4 for 25 single infections (~35.7%). Multiple OaPV coinfections were observed in 56 (~44.4%) positive samples. OaPV coinfections caused by two genotypes were observed in 31 positive samples (~55.4%), with dual OaPV3/OaPV4 infection being the most prevalent as seen in 11 blood samples. In addition, five OaPV1/OaPV4, four OaPV1/OaPV2, four OaPV2/OaPV3, four OaPV1/OaPV3, and three OaPV2/OaPV4 dual coinfections were also detected. OaPV coinfections by triple and quadruple genotypes were detected in 24 (~42.8%) and only one (~1.8%) of coinfected blood samples, respectively. Multiple infections caused by OaPV1/OaPV3/OaPV4 genotypes were the most prevalent, as observed in 12 (50%) blood samples harboring triple OaPV infections. This study showed that ddPCR is the most sensitive and accurate assay for OaPV detection and quantification thus outperforming real-time qPCR in terms of sensitivity and specificity. Therefore, ddPCR may represent the molecular diagnostic tool of choice, ultimately providing useful insights into OaPV molecular epidemiology and field surveillance.

Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

2020 ◽  
Author(s):  
Jyoti Kumar ◽  
G. G. Sonawane ◽  
Fateh Singh ◽  
S. Jegaveera Pandian ◽  
Rajiv Kumar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Bacterial Species ◽  
Sybr Green ◽  
Economic Losses ◽  
Conventional Pcr ◽  
Specific Detection ◽  
Pcr Assay ◽  
Tissue Samples

Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ µl of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P. multocida. The real-time PCR was found to be specific for KMT1 gene of P. multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P. multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.

Quantification and Differentiation of Campylobacter jejuni and Campylobacter coli in Raw Chicken Meats Using a Real-Time PCR Method

2007 ◽  
Vol 70 (9) ◽  
pp. 2015-2022 ◽  
Author(s):  
JOONBAE HONG ◽  
WOO KYUNG JUNG ◽  
JUN MAN KIM ◽  
SO HYUN KIM ◽  
HYE CHEONG KOO ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr ◽  
Campylobacter Species

Campylobacter species are one of the most common causes of bacterial diarrhea in humans worldwide. The consumption of foods contaminated with two Campylobacter species, C. jejuni and C. coli, is usually associated with most of the infections in humans. In this study, a rapid, reliable, and sensitive multiplex real-time quantitative PCR was developed for the simultaneous detection, identification, and quantification of C. jejuni and C. coli. In addition, the developed method was applied to the 50 samples of raw chicken meat collected from retail stores in Korea. C. jejuni and C. coli were detected in 88 and 86% of the samples by real-time quantitative PCR and the conventional microbiological method, respectively. The specificity of the primer and probe sets was confirmed with 30 C. jejuni, 20 C. coli, and 35 strains of other microbial species. C. jejuni and C. coli could be detected with high specificity in less than 4 h, with a detection limit of 1 log CFU/ml by the developed real-time PCR. The average counts (log CFU per milliliter) of C. jejuni or C. coli obtained by the conventional methods and by the real-time PCR assay were statistically correlated with a correlation coefficient (R2) between 0.73 and 0.78. The real-time PCR assay developed in this study is useful for screening for the presence and simultaneous differential quantification of C. jejuni and C. coli.

scholarly journals MOLECULAR DIAGNOSIS OF Guignardia citricarpa IN ASYMPTOMATIC SWEET ORANGE TISSUE

2017 ◽  
Vol 39 (4) ◽  
Author(s):  
FERNANDA DE SILLOS FAGANELLO ◽  
RENATO CARRER FILHO ◽  
VANESSA DUARTE DIAS ◽  
REGINA MELO SARTORI COELHO MORELLO ◽  
MARCOS GOMES DA CUNHA
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Black Spot ◽  
Leaf Tissue ◽  
Molecular Techniques ◽  
The European Union ◽  
Tissue Samples ◽  
Guignardia Citricarpa ◽  
Low Concentrations ◽  
Time Required

ABSTRACT Citrus black spot, a fungal disease caused by the quarantine fungus Guignardia citricarpa, restricts the exportation of fresh fruit to countries in the European Union. The occurrence of latent infections and the time required for diagnosis using conventional methods have brought about the need to validate fast, efficient and reproducible molecular techniques to detect the pathogen in asymptomatic tissue. As such, this study aims to detect G. citricarpa in the symptomatic fruit and asymptomatic leaf tissue of sweet oranges by conventional and real-time polymerase chain reaction (PCR). Specificity and limit of detection (LOD) were assessed in tissue samples of fruit lesions and asymptomatic leaves. Low concentrations of the fungus were found in asymptomatic leaves. Under these conditions, real-time PCR proved to be viable, reproducible and highly sensitive to detection of the pathogen.

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.

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