scholarly journals Development of a multiplex RT-PCR assay for simultaneous detection of Cucumber green mottle mosaic virus and Acidovorax citrulli in watermelon

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7539
Author(s):  
Xinyue Bi ◽  
Xiaodong Li ◽  
Haibo Yu ◽  
Mengnan An ◽  
Rui Li ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Citrullus Lanatus ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Transcriptional Elongation ◽  
Test Field ◽  
Rt Pcr ◽  
Internal Reference ◽  
Acidovorax Citrulli

Watermelon (Citrullus lanatus Thunb.) is considered as a popular and nutritious fruit crop worldwide. Watermelon blood flesh disease caused by Cucumber green mottle mosaic virus (CGMMV) and bacterial fruit blotch caused by Acidovorax citrulli, are two major quarantine diseases of watermelon and result in considerable losses to global watermelon production. In this study, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) method was developed for simultaneous detection of CGMMV and A. citrulli in both watermelon leaves and seeds. Two pairs of specific primers were designed based on the conserved sequences of the genomic RNA of CGMMV and the internal transcribed spacer of A. citrulli, respectively. Transcriptional elongation factor-1α from watermelon was added as an internal reference gene to prevent false negatives. No cross-reactivity was detected with other viral or bacterial pathogens infecting watermelon. Moreover, the multiplex RT-PCR showed high sensitivity and could simultaneously detect CGMMV and A. citrulli as little as 102 copies of plasmid DNA. This method was successfully applied to test field-collected watermelon leaves and stored seeds of cucurbitaceous crops. These results suggested that the developed multiplex RT-PCR technique is a rapid, efficient, and sensitive method for simultaneous detection of CGMMV and A. citrulli, providing technical support for monitoring, predicting, and preventing these two quarantine diseases. To our knowledge, this is the first report on simultaneous detection of a virus and a bacterium by multiplex RT-PCR in watermelon.

scholarly journals Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants

2021 ◽  
Author(s):  
Katarzyna Trzmiel
Keyword(s):  
Mosaic Virus ◽  
Simultaneous Detection ◽  
Brome Mosaic Virus ◽  
Mottle Virus ◽  
Grass Species ◽  
Replicase Gene ◽  
Rt Pcr ◽  
Pcr Products ◽  
Cereal Plants ◽  
Cocksfoot Mottle Virus

AbstractBrome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

scholarly journals EVALUATION OF ELEVEN IMMUNOCHROMATOGRAPHIC ASSAYS FOR SARS-CoV-2 DETECTION: INVESTIGATING DENGUE CROSS-REACTION

2020 ◽  
Author(s):  
Beatriz Araujo Oliveira ◽  
Lea Campos de Oliveira ◽  
Franciane Mendes de Oliveira ◽  
Geovana Maria Pereira ◽  
Regina Maia de Souza ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Diagnostic Techniques ◽  
Cross Reactivity ◽  
Cross Reaction ◽  
List Type ◽  
Rt Pcr ◽  
Serum Samples ◽  
Igm Antibodies ◽  
Good Agreement

AbstractBackgroundCOVID-19 disease (Coronavirus disease 2019) caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is widespread worldwide, affecting more than 11 million people globally (July 6th, 2020). Diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and low cost alternative for monitoring the spread of COVID-19 in the population.MethodsHere we evaluate the sensitivity and specificity of eleven different immunochromatographic tests in 98 serum samples from confirmed cases of COVID-19 through RT-PCR and 100 negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using 20 serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests.ResultsOur results demonstrated agreement between immunochromatographic assays and RT-PCR, especially after 10 days since the onset of symptoms. The evaluation of IgG and IgM antibodies combined demonstrated a strong level of agreement (0.85) of IC assays and RT-PCR. It was observed cross-reactivity between Dengue and COVID-19 using four different IC assays for COVID-19 diagnosis. The specificity of IC assays to detected COVID-19 IgM antibodies using Dengue serum samples varied from 80% to 85%; the specificity of IgG detection was 100% and total antibody was 95%.ConclusionsWe found high sensitivity, specificity and good agreement of IC assays, especially after 10 days onset of symptoms. However, we detected cross-reactivity between Dengue and COVID-19 mainly with IgM antibodies demonstrating the need for better studies about diagnostic techniques for these diseases.HighlightsImmunochromatographic assays demonstrated high sensitivity and specificity and good agreement with the gold-standard RT-PCR;Increase in sensitivity and specificity of assays using samples collected after the 10th day of symptoms;Cross-reaction with Dengue serology in evaluation of IgM.

scholarly journals Identification of Viruses Infecting Cigar and Flue-cured Tobacco Using Next Generation Sequencing and Establishment of Multiplex RT-PCR

2021 ◽  
Author(s):  
Tao Zhou ◽  
Shidong Zhou ◽  
Yong Chen ◽  
Jun Wang ◽  
Ruina Zhang ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Simultaneous Detection ◽  
Plant Viruses ◽  
Rt Pcr ◽  
Sequencing Platform ◽  
Agricultural Yield ◽  
And Control ◽  
Simultaneous Identification ◽  
Pooled Samples ◽  
Chilli Veinal Mottle Virus

Abstract Early, precise and simultaneous identification of the plant viruses is of great significance on preventing the spread of the viruses as well as reducing losses on agricultural yield. In this study, identification of plant viruses from symptomatic samples collected from cigar tobacco planting area in Deyang and flue-cured tobacco planting area in Luzhou city of Sichuan Province China was conducted by the deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform and plant virus specific contigs were generated based on the virus derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis was performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples with a total of 105 contigs being mapped to the closest plant viruses with the length range from 34~1720 nt. The results indicated that the major viruses were potato virus Y (PVY), Chilli veinal mottle virus (ChiVMV), tobacco vein banding mosaic virus (TVBMV), tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. These results provide theoretical basis and convenient methods for rapid detection and control of viruses on cigar and flue-cured tobacco.

Simultaneous detection of Apple Chlorotic Leaf Spot Virus and Apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR

2013 ◽  
Vol 164 ◽  
pp. 88-93 ◽  
Author(s):  
Santosh Watpade ◽  
Baswaraj Raigond ◽  
K.K. Pramanick ◽  
Neeraj Sharma ◽  
Anil Handa ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Leaf Spot ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Apple Rootstocks ◽  
Spot Virus ◽  
Chlorotic Leaf

scholarly journals Multiplex RT-PCR Reaction for Simultaneous Detection of Tomato Torrado Virus and Pepino Mosaic Virus Co-Infecting Solanum Lycopersicum

2013 ◽  
Vol 53 (3) ◽  
pp. 289-294 ◽  
Author(s):  
Przemysław Wieczorek ◽  
Aleksandra Obrępalska-Stęplowska
Keyword(s):  
Mosaic Virus ◽  
Solanum Lycopersicum ◽  
Internal Control ◽  
Production Efficiency ◽  
False Negative ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
High Resolution Melt ◽  
Pepino Mosaic Virus ◽  
Solanum Lycopersicum L

Abstract The tomato (Solanum lycopersicum L.) is cultivated all over the world and is a vegetable of significant economic importance. However, an increased production of the vegetable is directly connected with an elevated occurrence of pathogens limiting the production efficiency of the vegetable. Both, Tomato torrado virus and Pepino mosaic virus have been found to be serious disease factors. When not controlled, these viruses can significantly decrease tomato cultivation. In this article, we report a multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocol for simultaneous detection of both, Tomato torrado virus (ToTV) and Pepino mosaic virus (PepMV) in virus infected plants. The assay was designed to specifically amplify the conserved regions of genomic ribonucleic acid (RNA) of both viruses. Moreover, the glycerandehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control of amplification to exclude false-negative assay results. High-resolution melt analysis of generated RT-PCR products was additionally performed to increase sensitivity and double-check the specificity of the reaction without the need of subsequent complementary deoxyribonucleic acid (cDNA) sequencing

scholarly journals Identification of viruses infecting cigar and flue-cured tobacco using next generation sequencing and establishment of multiplex RT-PCR

2021 ◽  
Author(s):  
Tao Zhou ◽  
Shidong Zhou ◽  
Yong Chen ◽  
Jun Wang ◽  
Ruina Zhang ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Viruses ◽  
Simultaneous Detection ◽  
Plant Viruses ◽  
Rt Pcr ◽  
Sequencing Platform ◽  
Agricultural Yield ◽  
And Control ◽  
Simultaneous Identification ◽  
Pooled Samples

Abstract Backgrounds: Infection of plant viruses cause extensive damage to plants and reduce crop yield. Early, precise and simultaneous identification of the plant viruses is of great significance on preventing the spread of the viruses as well as reducing losses on agricultural yield.Methods: Identification of plant viruses from symptomatic samples collected from cigar tobacco planting area in Deyang and flue-cured tobacco planting area in Luzhou city of Sichuan Province China was conducted by the deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform and plant virus specific contigs were generated based on the virus derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis was performed to determine the species or strains of these viruses. Subsequently, specific primers were designed for simultaneous detection of five RNA viruses infecting tobacco.Results: A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples and a total of 105 contigs that can be mapped to the closest plant viruses with the length range from 34~1720 nt. The results indicated that the major viruses were potato virus Y (PVY), Chilli veinal mottle virus (ChiVMV), tobacco vein banding mosaic virus (TVBMV), tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). A fast and sensitive multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan.Conclusion: These results provide theoretical basis and convenient methods for rapid detection and control of viruses on cigar and flue-cured tobacco.

scholarly journals First Report of Cucumber mosaic virus Infecting Watermelon in Serbia

Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1706-1706 ◽  
Author(s):  
K. Milojević ◽  
I. Stanković ◽  
A. Vučurović ◽  
D. Ristić ◽  
D. Nikolić ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Citrullus Lanatus ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Total Rna ◽  
First Report ◽  
Cp Gene ◽  
Plant Pathol ◽  
Negative Controls

In June 2012, field-grown watermelon plants (Citrullus lanatus L.) with virus-like symptoms were observed in Silbaš locality, South Backa District of Serbia. Plants infected early in the growing season showed severe symptoms including stunting, mosaic, mottling, blistering, and leaf curling with reduced leaf size, while those infected at later stages exhibited only a mild mosaic. Affected plants were spread across the field and disease incidence was estimated at 40%. Thirteen symptomatic watermelon plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using a commercial diagnostic kit (Bioreba AG, Reinach, Switzerland) against the most important watermelon viruses: Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus (PRSV), and Squash mosaic virus (SqMV) (1). Commercial positive and negative controls and an extract from healthy watermelon tissue were included in each ELISA. Serological analyses showed that all plants were positive for CMV and negative for ZYMV, WMV, PRSV, and SqMV. The virus was mechanically transmitted from an ELISA-positive sample (449-12) to five plants of each Citrullus lanatus ‘Creamson sweet’ and Chenopodium amaranticolor using 0.01 M phosphate buffer (pH 7) with Serbian CMV isolate from Cucurbita pepo ‘Olinka’ (GenBank Accession No. HM065510) and healthy watermelon plants as positive and negative controls, respectively. Small necrotic lesions on C. amaranticolor and mild mosaic with dark green vein banding on watermelon leaves were observed on all inoculated plants 5 and 14 days post-inoculation, respectively. For further confirmation of CMV infection, reverse transcription (RT)-PCR was performed with the One-Step RT-PCR Kit (Qiagen, Hilden, Germany) using specific primers CMVCPfwd (5′-TGCTTCTCCRCGARWTTGCGT-3′) and CMVCPrev (5′-CGTAGCTGGATGGACAACCCG-3′), designed to amplify an 871-bp fragment of the RNA3 including the whole CP gene. Total RNA from 12 naturally infected and five mechanically infected watermelon plants was extracted with the RNease Plant Mini Kit (Qiagen). Total RNA obtained from the Serbian CMV isolate (HM065510) and healthy watermelon plants were used as positive and negative controls, respectively. The expected size of RT-PCR products were amplified from all naturally and mechanically infected watermelon plants but not from healthy tissues. The PCR product derived from isolate 449-12 was purified and directly sequenced using the same primer pair as in RT-PCR (JX280942) and analyzed by MEGA5 software (3). Sequence comparison of the complete CP gene (657 nt) revealed that the Serbian isolate 449-12 shared the highest nucleotide identity of 98.9% (99.1% amino acid identity) with the Spanish melon isolate (AJ829777) and Syrian tomato isolate (AB448696). To our knowledge, this is the first report of CMV on watermelon in Serbia. CMV is widely distributed within the Mediterranean basin where it has a substantial impact on many agricultural crops (2) and is often found to be prevalent during pumpkin and squash surveys in Serbia (4). The presence of CMV on watermelon could therefore represent a serious threat to this valuable crop in Serbia. References: (1) L. M. da Silveira et al. Trop. Plant Pathol. 34:123, 2009. (2) M. Jacquemond. Adv. Virus Res. 84:439, 2012. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vucurovic et al. Eur. J. Plant Pathol. 133:935, 2012.

scholarly journals Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257351
Author(s):  
Carolina de la Guardia ◽  
Giselle Rangel ◽  
Alcibiades Villarreal ◽  
Amador Goodridge ◽  
Patricia L. Fernández ◽  
...  
Keyword(s):  
Area Under The Curve ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Spike Protein ◽  
Close Relative ◽  
Rt Pcr ◽  
Characteristic Analysis ◽  
Serum Samples ◽  
Health Authorities ◽  
Significant Difference

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase–quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98–0.99). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24–0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92–0.99), specificity (range 0.93–0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87–0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus.

scholarly journals First Report of Cucumber green mottle mosaic virus on Melon in the United States

Plant Disease ◽  
2014 ◽  
Vol 98 (8) ◽  
pp. 1163-1163 ◽  
Author(s):  
T. Tian ◽  
K. Posis ◽  
C. J. Maroon-Lango ◽  
V. Mavrodieva ◽  
S. Haymes ◽  
...  
Keyword(s):  
United States ◽  
Mosaic Virus ◽  
Seed Production ◽  
Citrullus Lanatus ◽  
The United States ◽  
Rt Pcr ◽  
First Report ◽  
Rigid Rod ◽  
Yolo County ◽  
Das Elisa

In July 2013, a melon (Cucumis melo var. Saski) field in Yolo County, California, was inspected as part of a phytosanitary inspection for seed production. The leaves of the plants showed mosaic, green mottle, and blotches. When plant sap was examined using a transmission electron microscope, rigid rod-shaped particles were observed. Melon plant samples were analyzed by both CDFA and USDA APHIS PPQ laboratories and tested positive using DAS-ELISA against Cucumber green mottle mosaic virus (CGMMV) (Agdia, Elkhart, IN). To confirm the presence of CGMMV, total RNA was analyzed by RT-PCR using primers CGMMV-F5370 5′-CTAATTATTCTGTCGTGGCTGCGGATGC-3′ and CGMMV-R6390 5′-CTTGCAGAATTACTGCCCATA-3′ designed by PPQ based on 21 genomic sequences of CGMMV found worldwide. The 976-bp amplicon was sequenced (GenBank Accession No. KJ453559) and BLAST analysis showed the sequence was 95% identical to MP and CP region of CGMMV isolates reported from Russia (GQ495274, FJ848666), Spain (GQ411361), and Israel (KF155231), and 92% to the isolates from China (KC852074), Korea (AF417243), India (DQ767631), and Japan (D12505). These analyses confirm the virus was CGMMV. To our knowledge, this is the first report of CGMMV in the United States. Based on our sequence data, a second set of primers (CGMMV-F5796 5′-TTGCGTTTAGTGCTTCTTATGT-3′ and CGMMV-R6237 5′-GAGGTGGTAGCCTCTGACCAGA-3′), which amplified a 440-bp amplicon from CGMMV CP region, was designed and used for testing all the subsequent field and seed samples. Thirty-seven out of 40 randomly collected Saski melon samples tested positive for CGMMV, suggesting the virus was widespread in the field. All the melon samples also tested positive for Squash mosaic virus (SqMV) using DAS-ELISA (Agdia). Therefore, the symptoms observed likely resulted from a mixed infection. The melon field affected by CGMMV was immediately adjacent to fields of cucumber (Cucumis sativus var. Marketmore 76) and watermelon (Citrullus lanatus var. Sugar Baby) crops, both for seed production with no barrier between the crops. CGMMV was also detected from symptomatic plants from both fields. Seed lots used for planting all three crops were tested and only the melon seed was positive for CGMMV, suggesting the seed as the source of infection. The sequenced 440-bp RT-PCR amplicons from CGMMV-infected cucumber and watermelon plants and melon seeds were 99% identical to the CGMMV from the field melon. A cucumber plant infected with CGMMV but not SqMV was used for mechanical inoculation at the Contained Research Facility at University of California, Davis. Inoculated cucumber, melon, and watermelon plants showed green mottle and mosaic similar to that observed in the field. CGMMV is a highly contagious virus and damage by this virus on cucurbit crops has been reported in regions where CGMMV is present (2). CGMMV was detected on cucumber grown in greenhouses in Canada with 10 to 15% yield losses reported due to this virus (1). The three cucurbit crops in Yolo County were planted in an isolated area with no other cucurbits nearby. Measures, including destroying all the cucurbit plant material, have been taken to eradicate the virus. Use of CGMMV free cucurbit seed is necessary for prevention of this disease. References: (1) K.-S. Ling et al. Plant Dis. 98:701, 2014. (2) J. Y. Yoon et al. J. Phytopathol. 156:408, 2008.

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