scholarly journals Compared with conventional PCR assay, qPCR assay greatly improves the detection efficiency of predation

2020 ◽  
Vol 10 (14) ◽  
pp. 7713-7722 ◽  
Author(s):  
Ting‐bang Yang ◽  
Jie Liu ◽  
Jian Chen
Keyword(s):  
Detection Efficiency ◽  
Qpcr Assay ◽  
Conventional Pcr ◽  
Pcr Assay

Molecular detection and quantification of Xanthomonas albilineans in juice from symptomless sugarcane stalks using a real-time quantitative PCR assay

Plant Disease ◽  
2021 ◽  
Author(s):  
Yang Shi ◽  
Jian-Ying Zhao ◽  
Jing-Ru Zhou ◽  
Mbuya Sylvain Ntambo ◽  
Peng-Yuan Xu ◽  
...  
Keyword(s):  
Qpcr Assay ◽  
Taqman Probe ◽  
Bacterial Suspension ◽  
Bacterial Disease ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Leaf Scald ◽  
Sugarcane Production ◽  
Xanthomonas Albilineans ◽  
Detection And Quantification

Leaf scald, a bacterial disease caused by Xanthomonas albilineans (Ashby) Dowson, is a major limiting factor for sugarcane production worldwide. Accurate identification and quantification of X. albilineans is a prerequisite for successful management of this disease. A very sensitive and robust qPCR assay was developed in this study for detection and quantification of X. albilineans using TaqMan probe and primers targeting a putative adenosine triphosphate-binding cassette (ABC) transporter gene (abc). The novel qPCR assay was highly specific to the 43 tested X. albilineans strains belonging to different pulsed-field gel electrophoresis (PFGE) groups. The detection thresholds were 100 copies/µL of plasmid DNA, 100 fg/µL of bacterial genomic DNA, and 100 CFU/ml of bacterial suspension prepared from pure culture. This qPCR assay was 100 times more sensitive than a conventional PCR assay. The pathogen was detected by qPCR in 75.1% (410/546) symptomless stalk samples, whereas only 28.4% (155/546) samples tested positive by conventional PCR. Based on qPCR data, population densities of X. albilineans in symptomless stalks of the same varieties differed between two sugarcane production areas in China, Beihai (Guangxi province) and Zhanjiang (Guangdong province), and no significant correlation between these populations was identified. Furthermore, no relationship was found between these populations of the pathogen in asymptomatic stalks and the resistance level of the sugarcane varieties to leaf scald. The newly developed qPCR assay proved to be highly sensitive and reliable for the detection and quantification of X. albilineans in sugarcane stalks.

scholarly journals Use of "EP"(Peroxidase) allele in soybean varietal characterization

2014 ◽  
Vol 36 (4) ◽  
pp. 465-470
Author(s):  
Bárbara Panoff Valário ◽  
Cláudio Cavariani ◽  
José de Barros França-Neto ◽  
Elisa Serra Negra Vieira ◽  
Juliana Pereira Bravo ◽  
...  
Keyword(s):  
Plant Production ◽  
Pcr Analysis ◽  
Agarose Gel Electrophoresis ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Molecular Biology Technique ◽  
Soybean Cultivars ◽  
Colorimetric Test

The aim of this work was to evaluate the use of the molecular biology technique of PCR (Polimerase Chain Reaction) in the characterization of soybean cultivars. The study was performed at the Department of Plant Production, Faculty of Agricultural Sciences/ UNESP and Institute of Bioscience, Botucatu-SP. Fourteen commercial soybean cultivars were used, of which six were selected as positive reaction to peroxidase (BRS 320, BRS 284, BRS 232, BRS 7860RR, BRSMG 760SRR, BRS295RR), four as negative reaction (BRS 326, BRS 8160RR, BRSMG 800A (NutriSoy), BRS Valiosa RR) and four as double reaction (BRSGO 8060, BRS 270RR, FTS Campo Mourão and BRS 239). Thus, the results attained by the traditional biochemical colorimetric test for the 14 cultivars were compared with the conventional PCR assay. For PCR analysis, DNA was extracted from whole seeds and the primers were tested, and subsequently PCR and agarose gel electrophoresis were performed. The combination of primers prx9 + prx10 confirmed the use of the PCR reaction to characterize soybean cultivars considered doubtful by conventional colorimetric text.

scholarly journals Improved Primers for the Specific Detection of Leifsonia xyli subsp. xyli in Sugarcane Using a Conventional PCR Assay

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3251-3258
Author(s):  
Sheng-Ren Sun ◽  
Jun-Lü Chen ◽  
Yao-Yao Duan ◽  
Na Chu ◽  
Mei-Ting Huang ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Dna Amplification ◽  
High Yield ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Saccharum Spp ◽  
Sensitivity Tests ◽  
Field Samples ◽  
Pcr Assays ◽  
Higher Sensitivity

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx). Lxx infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel Lxx-specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published Lxx-specific primers and one universal Leifsonia generic primer pair LayF/LayR. Sugarcane leaf samples were collected from Saccharum spp. hybrids in commercial fields (315 samples) and from germplasm clones of five Saccharum species and Erianthus arundinaceus (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of Lxx genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural Lxx-infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse Leifsonia strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these Leifsonia strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and Lxx-pathogen detection with satisfactory sensitivity and specificity.

scholarly journals Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiyu Zhang ◽  
Ming Yao ◽  
Zhihui Tang ◽  
Daning Xu ◽  
Yan Luo ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Standard Deviation ◽  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
Simultaneous Detection ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Duck Tembusu Virus ◽  
Relative Standard

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

scholarly journals Detection and Genetic Analysis of Human Sapoviruses in River Water in Japan

2010 ◽  
Vol 76 (8) ◽  
pp. 2461-2467 ◽  
Author(s):  
Masaaki Kitajima ◽  
Tomoichiro Oka ◽  
Eiji Haramoto ◽  
Hiroyuki Katayama ◽  
Naokazu Takeda ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Real Time ◽  
River Water ◽  
Water Samples ◽  
Detection Efficiency ◽  
Water Environment ◽  
Filter Method ◽  
Rt Pcr ◽  
Pcr Assay ◽  
River Water Samples

ABSTRACT We investigated the prevalence of sapoviruses (SaVs) in the Tamagawa River in Japan from April 2003 to March 2004 and performed genetic analysis of the SaV genes identified in river water. A total of 60 river water samples were collected from five sites along the river, and 500 ml was concentrated using the cation-coated filter method. By use of a real-time reverse transcription (RT)-PCR assay, 12 (20%) of the 60 samples were positive for SaV. SaV sequences were obtained from 15 (25%) samples, and a total of 30 SaV strains were identified using six RT-PCR assays followed by cloning and sequence analysis. A newly developed nested RT-PCR assay utilizing a broadly reactive forward primer showed the highest detection efficiency and amplified more diverse SaV genomes in the samples. SaV sequences were frequently detected from November to March, whereas none were obtained in April, July, September, or October. No SaV sequences were detected in the upstream portion of the river, whereas the midstream portion showed high positive rates. Based on phylogenetic analysis, SaV strains identified in the river water samples were classified into nine genotypes, namely, GI/1, GI/2, GI/3, GI/5, GI/untyped, GII/1, GII/2, GII/3, and GV/1. To our knowledge, this is the first study describing seasonal and spatial distributions and genetic diversity of SaVs in river water. A combination of real-time RT-PCR assay and newly developed nested RT-PCR assay is useful for identifying and characterizing SaV strains in a water environment.

scholarly journals An Ultrafast One-Step qRT-PCR Assay for Detection of SARS-CoV-2

2021 ◽  
Author(s):  
Jadranka Milosevic ◽  
Mengrou Lu ◽  
Wallace Greene ◽  
Hong-Zhang He ◽  
Si-Yang Zheng
Keyword(s):  
Qpcr Assay ◽  
Nasopharyngeal Swab ◽  
Pcr Assay ◽  
Running Time ◽  
Clinical Sensitivity ◽  
Qrt Pcr ◽  
One Step

Abstract We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 mins on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

scholarly journals Multiplex PCR for Detection of Genes forStaphylococcus aureus Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin 1, and Methicillin Resistance

2000 ◽  
Vol 38 (3) ◽  
pp. 1032-1035 ◽  
Author(s):  
Manisha Mehrotra ◽  
Gehua Wang ◽  
Wendy M. Johnson
Keyword(s):  
Multiplex Pcr ◽  
Toxic Shock Syndrome ◽  
Methicillin Resistance ◽  
Positive Control ◽  
Toxic Shock ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Internal Positive Control ◽  
Toxic Shock Syndrome Toxin

A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (entA, entB,entC, entD, and entE), toxic shock syndrome toxin 1 (tst), exfoliative toxins A and B (etaA and etaB), and intrinsic methicillin resistance (mecA) was developed. Detection offemA was used as an internal positive control. The multiplex PCR assay combined the primers for sea tosee and femA in one set and those foreta, etb, tst, mecA, and femA in the other set. Validation of the assay was performed using 176 human isolates of Staphylococcus aureus. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.

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