Universal primers for rapid detection of six pospiviroids in Solanaceae plants using one-step RT-PCR and RT-LAMP

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are currently listed as quarantine pathogens in many countries. Among them, columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd) are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from one femtogram to 10 nanograms, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods currently in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate to screen a large number of solanaceous plants and seeds intended for import and export.

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Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

Plant Disease ◽

10.1094/pdis.1998.82.7.785 ◽

1998 ◽

Vol 82 (7) ◽

pp. 785-790 ◽

Cited By ~ 11

Author(s):

Vera L. A. Marinho ◽

J. Kummert ◽

G. Rufflard ◽

D. Colinet ◽

P. Lepoivre

Keyword(s):

Rt Pcr ◽

Degenerate Primers ◽

Apple Trees ◽

Active Growth ◽

Japanese Strain ◽

Apple Stem Grooving Virus ◽

Apple Stem ◽

Crude Extracts ◽

One Step ◽

The One

Partial nucleotide sequences of amplification products obtained from four European apple stem grooving virus (ASGV) isolates using degenerate primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Based on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were designed to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV isolates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to constitute good templates for reliable amplification of ASGV sequences from leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermore, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark tissues of apple trees during both active growth and the dormant season.

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Detection of avian influenza virus and newcastle disease virus by duplex one step RT PCR

Open Life Sciences ◽

10.2478/s11535-013-0164-7 ◽

2013 ◽

Vol 8 (6) ◽

pp. 520-526 ◽

Cited By ~ 1

Author(s):

Dawid Nidzworski ◽

Krzysztof Smietanka ◽

Zenon Minta ◽

Bogusław Szewczyk

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Egg Production ◽

Influenza Viruses ◽

Detection Methods ◽

Rt Pcr ◽

One Step

AbstractNewcastle disease Virus (NDV), a member of the Paramyxoviridae family, and Influenza virus, from the Orthomyxoviridae family, are two main avian pathogens that cause serious economic problems in poultry farming. NDV strains are classified into three major pathotypes: velogenic, mesogenic, and lentogenic. Avian influenza viruses (AIV) are also divided into: low pathogenic (LPAI) and highly pathogenic (HPAI) strains. Both viruses are enveloped, single stranded, negative-sense RNA viruses which give similar symptoms ranging from sub-clinical infections to severe disease, including loss in egg production, acute respiratory syndrome, and high mortality, depending on their level of pathogenicity. This similarity hinders diagnosis when based solely on clinical and post mortem examination. Most of the currently available molecular detection methods are also pathogenspecific, so that more than one RT-PCR is then required to confirm or exclude the presence of both pathogens. To overcome this disadvantage, we have applied a One Step Duplex RT-PCR method to distinguish between those two pathogens. The main objective of the project was to develop a universal, fast, and inexpensive method which could be used in any veterinary laboratory.

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Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids

10.1101/2020.04.17.046508 ◽

2020 ◽

Author(s):

Marleen Botermans ◽

Johanna W. Roenhorst ◽

Marinus Hooftman ◽

Jacobus Th.J. Verhoeven ◽

Eveline Metz ◽

...

Keyword(s):

Real Time ◽

Seed Transmission ◽

Potato Spindle Tuber Viroid ◽

Rt Pcr ◽

Tomato Seed ◽

Stunt Viroid ◽

Testing Laboratories ◽

Tomato Chlorotic Dwarf Viroid ◽

Development And Validation ◽

Pcr Test

AbstractPotato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the Pospisense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

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A broad spectrum, one-step RT-PCR to detect Satsuma dwarf virus variants using universal primers targeting both segmented RNAs 1 and 2

Journal of General Plant Pathology ◽

10.1007/s10327-011-0342-x ◽

2011 ◽

Vol 77 (6) ◽

pp. 326-330 ◽

Cited By ~ 11

Author(s):

Shin-ichi Shimizu ◽

Takao Ito ◽

Takanori Miyoshi ◽

Yasunobu Tachibana ◽

Tsutae Ito

Keyword(s):

Broad Spectrum ◽

Dwarf Virus ◽

Universal Primers ◽

Rt Pcr ◽

Satsuma Dwarf Virus ◽

One Step

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Evaluation of the Use of PCR and Reverse Transcriptase PCR for Detection of Pathogenic Bacteria in Biosolids from Anaerobic Digestors and Aerobic Composters

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4618-4627.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4618-4627 ◽

Cited By ~ 40

Author(s):

Carola Burtscher ◽

Stefan Wuertz

Keyword(s):

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Nucleic Acid ◽

Pathogenic Bacteria ◽

Organic Waste ◽

Detection Methods ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

One Step

ABSTRACT A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR- or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37°C and 24 h in Rappaport Vassiliadis medium at 43°C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples that tested positive when classical isolation procedures were followed. The study shows that selective two-step enrichment is useful when very low numbers of bacterial pathogens must be detected in organic waste materials, such as biosolids. There were no false-positive results derived from DNA of dead cells in the waste sample, suggesting that it is not necessary to perform RT-PCR analyses when PCR is combined with selective enrichment. Large numbers of added nontarget bacteria did not affect detection of Salmonella spp., L. monocytogenes, and Y. enterocolitica but increased the detection limit of Staphylococcus aureus from <10 to 104 CFU/g of organic waste. Overall, the detection methods developed using seeded organic waste samples from one waste treatment facility (WTF) needed to be modified for satisfactory detection of pathogens in samples from other WTFs, emphasizing the need for extensive field testing of laboratory-derived PCR protocols. A survey of 13 WTFs in Germany revealed that all facilities complied with the German Biowaste Ordinance, which mandates that the end product after anaerobic digestion or aerobic composting be free of Salmonella. In addition, all biosolids were free of L. monocytogenes, Staphylococcus aureus, and Y. enterocolitica, as evidenced by both classical and PCR-based detection methods.

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Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

10.1101/2020.03.12.988246 ◽

2020 ◽

Cited By ~ 12

Author(s):

Chenyu Li ◽

David N. Debruyne ◽

Julia Spencer ◽

Vidushi Kapoor ◽

Lily Y. Liu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Multiplex Pcr ◽

High Sensitivity ◽

Detection Methods ◽

Next Generation ◽

Rt Pcr ◽

Copy Numbers ◽

Virus Rna ◽

Highly Sensitive ◽

Generation Sequencing

AbstractMany detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, typically claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites. Therefore, alternative and highly sensitive methods are imperative. With the goal of improving sensitivity and accommodating various application settings, we developed a multiplex-PCR-based method comprised of 343 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens, and detected mutations through next generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel, that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates.

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First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) from Turkey and Germany

Plant Disease ◽

10.1094/pdis-93-3-0316a ◽

2009 ◽

Vol 93 (3) ◽

pp. 316-316 ◽

Cited By ~ 18

Author(s):

J. Th. J. Verhoeven ◽

M. Botermans ◽

J. W. Roenhorst ◽

J. Westerhof ◽

E. T. M. Meekes

Keyword(s):

Potato Spindle Tuber Viroid ◽

Rt Pcr ◽

Physalis Peruviana ◽

Source Of Infection ◽

Pcr Product ◽

Pcr Products ◽

Mother Plants ◽

Tomato Chlorotic Dwarf Viroid ◽

Plant Pathol ◽

Cape Gooseberry

Since the recent identification of Potato spindle tuber viroid (PSTVd) in vegetatively propagated ornamental plant species (4), many growers have asked to have their mother plants tested for this viroid. In December of 2007, a grower from Turkey submitted cuttings of cape gooseberry (Physalis peruviana) to be tested for PSTVd. Initial testing by real-time reverse transcription (RT)-PCR according to Boonham et al. (1) indicated the presence of either Mexican papita viroid, PSTVd, or Tomato chlorotic dwarf viroid in four samples. To identify the viroid(s) present, isolated RNA from these samples was used for RT-PCR (2), and products of the expected full genome size for the three viroids were amplified from each sample. One of the PCR products was sequenced (GenBank Accession No. EU862230) and analysis of the 357 nt sequence indicated it was most related to PSTVd sequences belonging to the so-called ‘Oceanian’ strain of the viroid (3), with 99.7% identity to GenBank Accession No. AY962324. Therefore, the viroid was identified as PSTVd. Pathogenicity of this PSTVd genotype was demonstrated when 4 weeks after mechanical inoculation with sap extracts seedlings of tomato cv. Money-maker showed the expected viroid symptoms of chlorosis and stunting, and the presence of the viroid in these plants was confirmed by RT-PCR (2). In March of 2008, by use of RT-PCR (2) and sequencing of the PCR product (GenBank Accession No. EU862231), PSTVd was identified in young seedlings of P. peruviana from a German grower. The German isolate differed at only three nucleotide positions from the Turkish isolate. The identification of PSTVd in young seedlings indicates that seeds had been source of infection, whereas in the case of the PSTVd infected cuttings from Turkey, the infection originated from infected mother plants. To our knowledge, these are the first reports of PSTVd in P. peruviana. Although infected P. peruviana plants did not show symptoms, they might act as sources of inoculum for crops like potato and tomato, which may suffer serious damage. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.

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