Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two<i> Badnavirus</i> Species in Sugarcane

Sun, Sheng‐Ren; Ahmad, Kashif; Wu, Xiao-Bin; Chen, Jiansheng; Fu, Hua‐Ying; Huang, Mei-Ting; Gao, San‐Ji

doi:10.1155/2018/8678242

Cited by 7 publications

(4 citation statements)

References 21 publications

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“…However, these assays are either laborintensive, less sensitive, require agarose gel analysis for the amplification products or had a risk of contamination, which may lead to false results. Real-time fluorescent quantitative PCR technology (qPCR) has become a powerful alternative platform for the detection and differentiation of pathogenic viruses [16][17][18][19][20]. In this study, a TaqMan based real-time fluorescent qPCR method, targeting the highly conserved DNA polymerase (DPOL) gene of PCMV, was developed for the rapid and reliable diagnosis of PCMV infection in porcine semen.…”

Section: Introductionmentioning

confidence: 99%

Development of a TaqMan Based Real-Time Fluorescent Quantitative PCR Assay for Detection of Porcine Cytomegalovirus in Semen

Chen

et al. 2020

BioMed Research International

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This study described a TaqMan based real-time fluorescent quantitative PCR (qPCR) method to detect porcine cytomegalovirus (PCMV) infection, targeting the conserved region of the DNA polymerase (DPOL) gene. The standard curve showed a linear regression relationship with a coefficient of 0.999 and a slope of y = −3.249x + 38.958 corresponding to the amplification efficiency at 99.8%. The limit of the qPCR method was 51.9 copies/μl. The established qPCR method showed excellent specificity, with no cross-reaction observed with common porcine pathogens. The coefficient of variation for intra-assay and interassay variability ranged up to 1.51% and 2.24%, respectively. PCMV positive signals can be found in semen using this qPCR method, which suggested that we should pay more attention to PCMV contamination in semen in order to eliminate PCMV infection in artificial insemination and xenotransplantation.

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Section: Introductionmentioning

confidence: 99%

Development of a TaqMan Based Real-Time Fluorescent Quantitative PCR Assay for Detection of Porcine Cytomegalovirus in Semen

Chen

et al. 2020

BioMed Research International

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“…In addition, companies such as Novateinbio and MyBioSource have ELISA products that detect antibody or antigen of PCMV, there are no commercialized products using molecular diagnostic methods until now. Real-time fluorescent quantitative PCR technology has become a powerful alternative platform for detection and differentiation of pathogenic viruses (3,17,18).…”

Section: Introductionmentioning

confidence: 99%

One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

Wang

Song²,

Shin³

et al. 2020

Front. Vet. Sci.

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Porcine cytomegalovirus (PCMV) is a pathogen that must be removed from pigs for use as organ donors in xenotransplantation. Recently, it has been found that when donor pigs are infected with PCMV, a pig-to-non-human-primate xenotransplantation lower transplant survival by 2-3 times. Therefore, highly sensitive methods are needed to maintain designated pathogen free (DPF) pig and screen for xenografts. The purpose of this study was to evaluate the performance of commercially available method with one-tube nested real-time PCR assay to quickly detect PCMV infection in clinical samples and compare the results with those of sequence analysis. Molecular diagnostic methods were used to evaluate 127 samples, including tissues and blood samples from pigs suspected of PCMV infection. The detection rate for positive PCMV was 38.6% (n = 49), 23.6% (n = 30), and 12.6% (n = 16) in one-tube nested real-time PCR, nested PCR, and conventional PCR methods, respectively. All PCMV-positive samples in conventional PCR or nested PCR methods were also positive in the one-tube nested real-time PCR assay. All the PCR products in the three methods were checked for amplification of PCMV gene by PCR and subsequent direct sequencing. The results of one-tube nested real-time PCR were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). Our study found that the one-tube nested real-time PCR assay is more sensitive than the other two methods. This assay required approximately 1.5 h for completion. Therefore, we concluded that one-tube nested real-time PCR assay is a fast and reliable method for the characterizing pathogen responsible for PCMV infection.

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“…This amiRNA-based silencing strategy was applied effectively in many plants to combat plant viruses: cotton leaf curl Kokhran virus (CLCuKoV-Bu) [19], Cucumber mosaic virus (CMV) [20], CymMV and ORSV [21]. Serological method, ELISA, immunosorbent electron microscopy (ISEM) [22], IC-PCR [23], RT-PCR [24], rolling circle amplification (RCA) [25] are the most widely used reliable detection techniques for SCBV. New strategies are under development for improved diagnoses and cure of badnaviruses [26].…”

Section: Introductionmentioning

confidence: 99%

An Algorithmic framework for genome-wide identification of Sugarcane (Saccharum officinarumL.)-encoded microRNA targets against SCBV

Ashraf¹,

Feng

Ashraf

et al. 2020

Preprint

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Sugarcane Bacilliform Virus (SCBV) is considered an economically the most damaging pathogen for sugarcane production worldwide. Three ORFs are characterized in a single molecule of circular, ds-DNA genome of the SCBV, encoding for hypothetical protein (ORF1), DNA binding protein (ORF2) and Polyprotein (ORF3). The study was aimed to predict and comprehensively evaluate sugarcane miRNAs for the silencing of SCBV genome using in-silico algorithms. Computational methods were used for prediction of candidate miRNAs from sugarcane (S. officinarum L.) to silence the expression of SCBV genes through translational inhibition by mRNA cleavage. Mature sugarcane miRNAs were retrieved and were assessed to hybridization with the SCBV genome. A total of fourteen potential candidate miRNAs from sugarcane were computed by all the algorithms used for the silencing of SCBV. A consensus of three algorithms predicts hybridization sites of sof-miR159e at common locus 5534. The miRNA-mRNA interaction was estimated by computing free-energy of miRNA-mRNA duplex using RNAcofold algorithm. Regulatory network of predicted candidate miRNAs of sugarcane with SCBV ORFs, generated using Circos, identify novel targets. Consequently, detecting and discarding inefficient amiRNAs prior to cloning would help suppressed mutants faster. The efficacy of predicted candidate miRNAs was evaluated to test the survival rate of the in vitro amiRNA-mediated effective badnaviral silencing and resistance in sugarcane cultivars.

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

Cited by 7 publications

References 21 publications

Development of a TaqMan Based Real-Time Fluorescent Quantitative PCR Assay for Detection of Porcine Cytomegalovirus in Semen

Development of a TaqMan Based Real-Time Fluorescent Quantitative PCR Assay for Detection of Porcine Cytomegalovirus in Semen

One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

An Algorithmic framework for genome-wide identification of Sugarcane (Saccharum officinarumL.)-encoded microRNA targets against SCBV

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