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Development of PCR diagnostic system for detection of the seed-transmitted tobacco ringspot virus in quarantine.

ABSTRACT: Tobacco ringspot virus (TRSV) is a plant quarantine virus in Korea. As such, a TRSV examination is conducted when importing various crops. In this study, RT-PCR and nested PCR systems for TRSV detection in quarantine sites, and the modified-positive control plasmid for proving laboratory contamination and false positive reactions were developed. The developed diagnostic system was used to detect TRSV in the quarantine site. It revealed that from 2012 to August 2014, a total of 12 cases were detected in imported various crops. The system is expected to continue contributing to TRSV detection in plant quarantine.

SUBMITTER: Lee S

PROVIDER: S-EPMC4363257 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Development of PCR diagnostic system for detection of the seed-transmitted tobacco ringspot virus in quarantine.

Lee S S Lee G G Choi I C IC Rho J Y JY

Indian journal of microbiology 20150225 2

Tobacco ringspot virus (TRSV) is a plant quarantine virus in Korea. As such, a TRSV examination is conducted when importing various crops. In this study, RT-PCR and nested PCR systems for TRSV detection in quarantine sites, and the modified-positive control plasmid for proving laboratory contamination and false positive reactions were developed. The developed diagnostic system was used to detect TRSV in the quarantine site. It revealed that from 2012 to August 2014, a total of 12 cases were dete ...[more]

PMID: 25805911

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Project description:Papaya ringspot virus (PRSV) is a significant threat to global papaya cultivation, causing ringspot disease, and it belongs to the species Papaya ringspot virus, genus Potyvirus, and family Potyviridae. This study aimed to assess the occurrence and severity of papaya ringspot disease (PRSD) in major papaya-growing districts of Karnataka, India, from 2019 to 2021. The incidence of disease in the surveyed districts ranged from 50.5 to 100.0 percent, exhibiting typical PRSV symptoms. 74 PRSV infected samples were tested using specific primers in RT-PCR, confirming the presence of the virus. The complete genome sequence of a representative isolate (PRSV-BGK: OL677454) was determined, showing the highest nucleotide identity (nt) (95.8%) with the PRSV-HYD (KP743981) isolate from Telangana, India. It also shared an amino acid (aa) identity (96.5%) with the PRSV-Pune VC (MF405299) isolate from Maharashtra, India. Based on phylogenetic and species demarcation criteria, the PRSV-BGK isolate was considered a variant of the reported species and designated as PRSV-[IN:Kar:Bgk:Pap:21]. Furthermore, recombination analysis revealed four unique recombination breakpoint events in the genomic region, except for the region from HC-Pro to VPg, which is highly conserved. Interestingly, more recombination events were detected within the first 1710 nt, suggesting that the 5' UTR and P1 regions play an essential role in shaping the PRSV genome. To manage PRSD, a field experiment was conducted over two seasons, testing various treatments, including insecticides, biorationals, and a seaweed extract with micronutrients, alone or in combination. The best treatment involved eight sprays of insecticides and micronutrients at 30-day intervals, resulting in no PRSD incidence up to 180 days after transplanting (DAT). This treatment also exhibited superior growth, yield, and yield parameters, with the highest cost-benefit ratio (1:3.54) and net return. Furthermore, a module comprising 12 sprays of insecticides and micronutrients at 20-day intervals proved to be the most effective in reducing disease incidence and enhancing plant growth, flowering, and fruiting attributes, resulting in a maximized yield of 192.56 t/ha.

Project description:A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus ORTHOPOXVIRUS: A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (T(m)s) determined for 46 strains of variola virus (T(m)s, 55.9 to 57.8 degrees C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (T(m)s, 61.7 to 62.7 degrees C), 15 strains of monkeypox virus (T(m)s, 61.9 to 62.2 degrees C), 40 strains of cowpox virus (T(m)s, 61.3 to 63.7 degrees C), 8 strains of mousepox virus (T(m), 61.9 degrees C), and 8 strains of camelpox virus (T(m)s, 64.0 to 65.0 degrees C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.

Dataset Information

Development of PCR diagnostic system for detection of the seed-transmitted tobacco ringspot virus in quarantine.

Publications

Development of PCR diagnostic system for detection of the seed-transmitted tobacco ringspot virus in quarantine.

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