Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR

Singh, R. P.; Nie, Xianzhou; Singh, Mathuresh; Coffin, Robert S.; Duplessis, P.

doi:10.1016/s0166-0934(01)00391-3

Cited by 72 publications

(41 citation statements)

References 28 publications

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“…This problem could be avoided using commercial kits for purification of nucleic acids, but their cost is prohibitive for detecting the virus in epidemiological studies due to the large number of samples that need to be processed. These inhibitors are very common in plants, but do not affect molecular hybridization procedures (Singh et al, 2002). Tissue-print hybridization was consistent and permitted detection of PMoV-T on imprints from all infected tomato plants collected from the appropriate selected material, and turned out to be as robust as dot-blot hybridization.…”

Section: Discussionmentioning

confidence: 82%

Detection of a tomato strain of Parietaria mottle virus (PMoV‐T) by molecular hybridization and RT‐PCR in field samples from north‐eastern Spain

et al. 2005

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Parietaria mottle virus (PMoV-T) infecting tomatoes from different commercial fields in Catalonia, Spain was detected by dot-blot and tissue-printing hybridization and one-step reverse transcriptase PCR (RT-PCR). Specific cDNA was amplified by RT-PCR from mechanically infected Chenopodium quinoa plants and cloned into a plasmid vector. A digoxigenin-labelled riboprobe was synthesized for molecular hybridization, and a specific set of primers (PMoV-1F/ PMoV-1R) were designed for one-step RT-PCR assays. Material from different parts of PMoV-T-infected tomato plants was used for the analysis. The virus was detected on necrotic apical shoots, fruits with symptoms, and symptomless shoots grown from old necrotic branches, but not on the old necrotic branches themselves. Both molecular hybridization and one-step RT-PCR procedures were highly specific, and no cross-reactions were observed with other related ilarviruses. PMoV-T was detected successfully by molecular techniques on all infected tomato plants collected from commercial fields, although one-step RT-PCR was less robust than molecular hybridization. The observed reliability of tissue-printing hybridization could greatly facilitate both the large-scale analysis and epidemiological studies of this important pathogen.

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Section: Discussionmentioning

confidence: 82%

Detection of a tomato strain of Parietaria mottle virus (PMoV‐T) by molecular hybridization and RT‐PCR in field samples from north‐eastern Spain

et al. 2005

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“…Due to the advantages of RT-PCR system (single/multiplex) concerning on time, cost, reliability and sensitivity, it has been documented as an alternative system for certification of plant propagating materials. Disadvantages of this system including the presence of inhibitors such as polyphenolics, polysaccharides and endogenous ribonucleases in the plant extracts (20) and inefficient synthesis of viroid genome (3) could be easily removed by proper RNA extraction methods and using proper sequence primers. Moreover, the accuracy and reliability of the extracted RNA and subsequently cDNA synthesis could be analyzed by the coamplification of the internal control (Nad 5 gene) in both d-and m-RT PCR.…”

Section: Discussionmentioning

confidence: 99%

Development of a Multiplex RT-PCR Assay for Detection of the Causal Agents of Citrus Tristeza and Cachexia Diseases with Coamplification of Plant mRNA as an Internal Control

et al. 2011

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Background and Aims: Plant certification programs need reliable, fast, cheap and sensitive methods for detection of systemic pathogens with special interest in virus and viroid detection. Reverse transcriptase-polymerase chain reaction (RT-PCR) has been documented as an alternative assay for certification of plant propagating materials. The main object of the present study was the optimization of a multiplex RT-PCR assay for simultaneous detection of Citrus tristeza virus (CTV) and Hop stunt viroid (HSVd), the casual agents of citrus tristeza and cachexia, together with the plant mRNA as an internal control for citrus certification in the country. Materials and Methods: Total RNA was extracted from healthy; CTV-and HSVd-infected citrus tissues and subjected to cDNA synthesis by M-MuLV H -reverse transcriptase, followed by optimization of simplex, diplex and multiplex RT-PCR (s-, d-, and mRT-PCR). Amplified fragments were further sequenced for evaluation of the accuracy of the assays. Results: CTV, HSVd and the plant internal control (Nad 5 gene) were successfully amplified in all assays and the sequence information revealed the accuracy of all assays in citrus certification programs. Conclusion:Results from the developed s-, d-, and mRT-PCR assays revealed these detection methods as excellent candidates for citrus certification.

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“…Samples were stored at 4°C. Various other DNA extraction protocols were used on plant samples to remove inhibitors [20][21][22][23][24], including the PowerPlant Pro DNA isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA).…”

Section: Dna Extractionmentioning

confidence: 99%

Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

Yasuhara-Bell¹,

Ayin²,

Hatada³

et al. 2015

J Plant Pathol Microbiol

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Klebsiella spp. are opportunistic pathogens with clinical, veterinary and plant-associated isolates. A previous study showed that bacterial ooze from wetwood of severely declined ironwood trees in Guam contained Ralstonia solanacearum, Klebsiella variicola and K. oxytoca. In this study, Loop-Mediated Isothermal Amplification (LAMP) detected K. variicola and K. oxytoca specifically, using unique primer sets designed individually for each organism. Each LAMP detected its target specifically, while showing negative results for non-target bacteria and negative controls. LAMP detected Klebsiella in inoculated-ironwood stem tissues and bacterial ooze. Due to the presence of plant inhibitors, different sampling protocols were tested. Soaking plant tissue samples to allow diffusion of bacteria into solution, followed by boiling, provided optimum detection of Klebsiella directly from plant samples. False negatives obtained when using crushed plant samples were eliminated by including an enrichment step, involving plating and 12-h incubation. DGGE (denaturing gradient gel electrophoresis) and a colony-blot immunoassay using a Klebsiella-specific antibody also detected Klebsiella in inoculated ironwood. DGGE bands and antibody crossreactions from closely related enterobacters showed the potential for false positive results. The nature of LAMP makes it ideal for point-of-care testing, and when combined with the specificity of the LAMP primers developed in this study, demonstrates its potential as a routine field test for Klebsiella in ironwood in Guam, as well as clinical and veterinary diagnosis of Klebsiella infection. Additionally, the regions targeted for detection in this study have application across all forms of molecular-based diagnostics.

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Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR

Cited by 72 publications

References 28 publications

Detection of a tomato strain of Parietaria mottle virus (PMoV‐T) by molecular hybridization and RT‐PCR in field samples from north‐eastern Spain

Detection of a tomato strain of Parietaria mottle virus (PMoV‐T) by molecular hybridization and RT‐PCR in field samples from north‐eastern Spain

Development of a Multiplex RT-PCR Assay for Detection of the Causal Agents of Citrus Tristeza and Cachexia Diseases with Coamplification of Plant mRNA as an Internal Control

Specific Detection of Klebsiella variicola and K. oxytoca by Loop-Mediated Isothermal Amplification

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