Development of real-time fluorescent quantitative PCR assay for detection of PRRSV based on TaqMan probe
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We establish a TaqMan real-time PCR assay for detection of PRRSV.The specific primers and probes were designed in the conserved region of the ORF7 gene for PRRSV,and the real-time fluorescent quantitative PCR was established by optimizing the probe concentration.Thirty clinical samples were detected by using the established quantitative RT-PCR assay,and the results were compared with that of conventional RT-PCR and viral isolation tests.TaqMan fluorescent quantitative PCR for detection of PRRSV was established successfully with the optimal probe concentration 0.4 μmol,and detection limit was as low as 3.51 copies/μl.The results by the TaqMan real-time PCR method were 100% consistent with the viral isolation tests.Sensitivity and positive rate(28/30) for clinical samples of TaqMan fluorescent quantitative PCR were relatively higher than conventional PCR(25/30).The results indicated this method has high specificity,sensitivity and reproducibility,and could be used for the diagnosis of PRRSV infection.Keywords:
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The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.
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To design and rapidly evaluate a TaqMan assay for detecting influenza A viruses.The probe and the primers of the assay were designed with the software packages of DNA Star and Primer Premier 5.0. Their specificity and conservation were verified through Blast in GenBank and electronic hybridization. The assay's sensitivity was compared with the standard RT-PCR.The designed primers and probe were confirmed to be very specific and conserved. The assay was 3-27 folds more sensitive than the standard RT-PCR. The RT and PCR steps could be simplified into one step.The TaqMan Real-time PCR assay is specific, sensitive and easy to perform.
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A pair of primers and a TaqMan probe were designed and synthesized according to the conserved gene sequences of Infectious Hypodermal and Haematopoietic Necrosis(IHHNV)in GenBank(AF218226),and then reaction parameters were optimized to develop a real-time TaqMan-quantitative PCR assay.The developed quantitative PCR assay was compared with that of routine PCR.This quantitative PCR assay could detect 2 template copies of plasmid DNA,and its sensitivity was 1 000 times higher than that of the routine PCR.The real-time TaqMan-quantitative PCR results of 15 routine PCR positive clinical samples showed that concentration of the clinical samples were 2.15×107~4.21×104 copies/μL.The samples were examined using the quantitative PCR repeatedly and the results indicated that the quantitative PCR was reproducible and could be used successfully for the diagnosis of IHHNV infection.
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Objective In order to develop a highly specific and sensitive real-time PCR assay to detect and indentify Anaplasma,the agent of Anaplasmoses. Methods A specific msp-2 outer membrane protein gene of A.phagocytophilum was selected for designing general TaqMan probe and TaqMan MGB probe based on the conserved sequences of msp-2 genes of 39 isolates all over the world. The evaluation of assay included the specificity,lowest detection load (LOD) and stability. Results Similar specificity and reproducibility for the two real-time PCR methods with general TaqMan probe and TaqMan MGB probe were demonstrated. Besides A.phagocytophilum,17 members of Rickettsia and other 8 common pathogens in clinical hospitals were not amplified. The LOD by PCR with TaqMan MGB probe was 10 times than that by PCR with general TaqMan. Conclusion Two highly specific and sensitive real-time PCR assays were developed to test A.phagocytophilum infection during the early phase of illness,but the assay with TaqMan MGB probe is more sensitive than that with TaqMan probe.
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Anaplasma phagocytophilum
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According to the human GABRA1 gene sequences available in GenBank,a pair of primers and a TaqMan probes are designed to establish a TaqMan probe based real-time PCR method for GABRA1 gene of human.To eatablish the standard curve,the product of conventional PCR is served as a standard.The result shows the linear range of Ct value is from 12.07 to 32.72 with a good correlation coefficient(r=0.999).The real-time PCR assay developed in this study can detect GABRA1 in expanded range with high efficiency,less time and sensitiveness.
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A Taqman real-time fluorescence quantitative assay for the rapid detection of milk allergenα-lactalbumin in food was established.PCR primers and Taqman probe were designed based on the gene sequence ofα-lactalbumin(GenBank No.AF249896.1)for real-time PCR.Plasmids were prepared as the standard PCR template and identified with enzyme digestion and then were subjected to sequencing.Then real-time fluorescence PCR assay was performed.Theα-lactalbumin DNA fragment has been cloned successfully and the standard curve had a good linear relationship ranging from 1.12×103 to 1.12×108copies.The detection sensitively of liquid sample reached up to 1 000copies/mL.Furthermore,the specificity and stability of the method were good.The real-time PCR method has been developed successfully and it could be applied toα-lactalbumin detection with high sensitivity and specificity.
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A real-time fluorescent TaqMan-quantitative PCR assay using 2 pairs of primers and a probe designed and synthesized according to the P32 gene of the virus genome was developed for the specific detection of capripox virus in epithelial suspensions and cell culture preparations. The real-time fluorescent PCR assay detected specifically CaPV virus in samples with greater sensitivity than the conventional PCR procedure. The fluorescent PCR assay was fast, and could quantitatively assess the virus amounts and could handle more samples and/or replicates of samples in a single assay than the conventional PCR procedure. The results showed that this assay is a valuable complementary tool to the routine diagnostic procedures for the detection of capripox virus infection.
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Assay for the detection and quantification of porcine circovirus type 2 (PCV 2) with the real-time PCR were developed. TaqMan probe real-time using a set of primer/probe was developed for detection of PCV 2. In this study we applied real-time PCR assay to 320 samples, collected from pig farms. In 151 of 320 samples, PCV 2 DNA was detected by conventional PCR assay. All samples positive for PCV 2 DNA in conventional PCR assay were also positive in Real-time PCR assay, but 69 of 169 samples that tested negative for PCV 2 DNA in conventional assay were tested positive in TaqMan probe real-time PCR assay. The test of TaqMan probe real-time PCR resulted in detection and quantification limits of 101 copies per sample. TaqMan probe real-time PCR assay increased the number of samples in which PCV 2 was detected by 21%. TaqMan probe real-time PCR assay is very efficient method in contrast to the conventinal PCR, becoming increasingly important method for gene analysis.
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The primers and probes were designed and synthesized according to the conserved VP1 sequences of porcine Sapovirus(SaV),and a TaqMan fluorescence quantitative RT-PCR assay were developed by optimizing the reaction conditions.Results showed that the fluorescence quantitative RT-PCR assay could detect 16.1 copies·μL-1 of plasmid DNA,while the sensitivity of the routine RT-PCR was 1.61 ×103 copies·μL-1.216 stool samples were then detected by the established quantitative RT-PCR assay,and the results were compared with that of routine RT-PCR.It also showed that the sensitivity of established method was higher than that of the routine RT-PCR.Phylogenetic analysis indicated that all of the 4 SaV strains we had identified belonged to GⅢ,and shared 100% nucleotide homology with another Shanghai porcine SaV strain(FJ387164).The TaqMan fluorescence quantitative PCR assay,which is more specific,sensitive and accurate,can be used for the epidemiological investigation and diagnosis of porcine SaV infection.
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