Use of Unique RNA Sequence-Specific Oligonucleotide Primers for RT-PCR to Detect and Differentiate Soybean Mosaic Virus Strains

Omunyin, M. E.; Hill, John H.; Miller, W. Allen

doi:10.1094/pd-80-1170

Cited by 15 publications

(3 citation statements)

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“…Since these viruses have only recently been recognized, descriptions of further CBSD causing viruses can be expected (Ndunguru et al, 2015). Robertson et al (1991) and Omunyin et al (1996) stated that based on the nucleotide sequence information of several different viruses, specific primers are designed and used in PCR to detect and differentiate viruses at the family, genus, or strain level. Also, simultaneous detection of unrelated viruses in a sample by using a mixture of virus-specific primer is possible ("Multiplex" PCR) (Bariana et al, 1994;Abarshi et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Attempts to Identify Cassava Brown Streak Virus in Western Democratic Republic of Congo

Bakelana¹,

Magembe²,

Laura³

et al. 2019

JAS

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Root necrosis similar to those of the cassava brown streak disease (CBSD) were observed on cassava in western provinces of the Democratic Republic of Congo (DR.Congo) in the early 2000’s. However molecular laboratory diagnosis were not able to detect any causative agent responsible for the attacks, hence, the disease related to these symptoms was named CBSD-like disease. In order to assess the distribution and the incidence of the CBSD-like disease, surveys were carried out in four western provinces, comprising, Kwango and Kwilu, Sud Ubangi, Kinshasa and Kongo Central. CBSD-like disease was observed in all surveyed provinces on the basis of root symptoms because foliar symptoms were different to those of the documented cases of CBSD in other parts of east Africa. CBSD-like disease incidence was high in Kongo Central and Sud Ubangi, exceeding an average of 50 %, but low in Kwango and Kwilu (32.8%) and in Kinshasa (19.1%). During the surveys, cassava leaf samples were collected for lab identification of the causal agent. PCR diagnosis was done on these samples using primers specific for the two known CBSVs. All samples tested negative with no amplification of DNA fragments of the correct size.  Thus, further analysis on the causative organism is needed using Next Generation Sequencing (NGS) approaches. NGS approaches will help also to identify the causative organism in other Central Africa countries (Angola, Congo-Brazzaville and Gabon) where such cassava root necrosis have been reported or are suspected.

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Section: Discussionmentioning

confidence: 99%

Attempts to Identify Cassava Brown Streak Virus in Western Democratic Republic of Congo

Bakelana¹,

Magembe²,

Laura³

et al. 2019

JAS

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“…The complete nucleotide sequences of SMV strain G2 and G7 have been determined (8) and most of the variation was observed in P1, helper component-protease (HC-Pro), P3, and cylindrical inclusion (CI) regions. Omunyin et al (15) first used RT-PCR assay to detect and discriminate two SMV strains, G2 and G7. They have shown that unique RNA-specific primers can be employed to detect and discriminate between RNAs of very similar strains.…”

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confidence: 99%

Identification of Soybean mosaic virus Strains by RT-PCR/RFLP Analysis of Cylindrical Inclusion Coding Region

Kim

Kim²,

Roh

et al. 2004

Plant Disease

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A reverse-transcriptase polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) was employed successfully for detection and identification of Soybean mosaic virus (SMV) strains. A primer pair amplifying a 1,385-bp fragment of the cylindrical inclusion (CI) coding region was used for RT-PCR and the RFLP profiles of the RT-PCR products were compared after restriction digestion with RsaI, EcoRI, or AccI restriction endonucleases. These enzymes were chosen based on the nucleotide sequences of SMV strains G2, G5, G5H, G7, and G7H in the CI coding region. These five strains, as well as seedborne SMV isolates from local soybean cultivars, could be differentiated by RT-PCR/RFLP analysis. The results correlated well with strain identification by symptom phenotypes produced on differential cultivars inoculated with strains and isolates. The sensitivity of RT-PCR enabled detection of SMV from plants with necrotic symptoms in which the number of virus particles was too low to be detected by enzyme-linked immunosorbent assay.

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“…Use of PCR has been widely applied for the rapid detection of viruses and viroids (11). Strain-specific primers were used in PCR and nucleic acid hybridization techniques for strain differentiation of citrus tristeza closterovirus (3) and soybean mosaic potyvirus (13). The inability of native Taq DNA polymerase to correct mismatches between the template DNA and the 3′ end of the primer was utilized in developing strain-specific primers for necrotic (PStV-Ts) and non-necrotic (PStV-B and PStV-IB) isolates of PStV.…”

Section: Resultsmentioning

confidence: 99%

Differentiation of Biologically Distinct Peanut Stripe Potyvirus Strains by a Nucleotide Polymorphism-Based Assay

Pappu¹,

Pappu

Chang

et al. 1998

Plant Disease

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A necrotic strain of peanut stripe potyvirus (PStV-Ts) was used to design and test strain-differentiating oligonucleotides. The 3′ region of PStV-Ts, including a part of the NIb region, the complete coat protein (CP) gene, and the 3′-untranslated region, was cloned and sequenced. PStV-Ts had a high degree of sequence identity (92 to 95%) to the known non-necrotic (blotch) strains both at the nucleotide and amino acid sequence levels. Nucleotide sequence differences unique to the necrotic strain were identified when compared to the available non-necrotic isolates of PStV. Nucleotide polymorphism in the CP gene sequences was utilized in designing oligonucleotides that were specific to the necrotic strain, and were employed in an assay to differentiate the necrotic strain from non-necrotic. The 3′ end mismatch in the oligonucleotides contributed in particular to the differentiation of the strains. This approach facilitated rapid, sensitive, and reliable detection and differentiation of PStV strains.

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Use of Unique RNA Sequence-Specific Oligonucleotide Primers for RT-PCR to Detect and Differentiate Soybean Mosaic Virus Strains

Cited by 15 publications

References 0 publications

Attempts to Identify Cassava Brown Streak Virus in Western Democratic Republic of Congo

Attempts to Identify Cassava Brown Streak Virus in Western Democratic Republic of Congo

Identification of Soybean mosaic virus Strains by RT-PCR/RFLP Analysis of Cylindrical Inclusion Coding Region

Differentiation of Biologically Distinct Peanut Stripe Potyvirus Strains by a Nucleotide Polymorphism-Based Assay

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