A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2)

Dey, Kishore; Lin, Hong; Borth, Wayne B.; Melzer, Michael; Hu, John

doi:10.1016/j.jviromet.2012.03.025

Cited by 9 publications

(12 citation statements)

References 15 publications

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“…A simple colorimetric technique using gold nanoparticles in LAMP reactions to detect PMWaV-1 and PMWaV-2 has also been developed, which takes 40 min to complete and has a detection limit of 10 copies of viral DNA. Another novel technique, single-tube dual primer-PCR (STDP-PCR) using nested PCR primers has been developed that can detect very low titers of PMWaV-2 that are below the levels detected with RT-PCR [68].…”

Section: Detection Of Pmwavsmentioning

confidence: 99%

“…PMWaV-1 and -3 both have seven open reading frames, but PMWaV-1 lacks the intergenic region that occurs between RdRp and p6 ORFs in PMWaV-3, and the CPd ORF [57]. The PMWaV-3 genome sequence encompasses 11891 nt, and encodes seven ORFs and the untranslatable region at the 3'-end [55,68]. PMWaV-3 lacks an intergenic region between ORFlb and ORF2 that is present in PMWaV-2, and encodes a 28.8 kDa coat protein but lacks the coat protein duplicate (CPd) found in PMWaV-2.…”

Section: Pmwav-1 and -3mentioning

confidence: 99%

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Mealybug Wilt of Pineapple and Associated Viruses

et al. 2018

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Mealybug wilt of pineapple (MWP) is a disease of pineapple that has a long history in Hawaii, but is present throughout the world where pineapples are grown in tropical regions. The disease has an interesting etiology that is poorly understood but involves an association with virus particles, mealybug vectors, and ants which spread the mealybug vectors. Several distinct pineapple mealybug wilt-associated virus (PMWaV) species have been identified thus far with potential further member species yet to be characterized. Pineapple mealybug wilt-associated viruses are member species of the Ampelovirus genus of the Closteroviridae family. Ampeloviruses are split into two subgroups, subgroup I and subgroup II. PMWaV-2 is a subgroup II member, and these have a longer and more complex genome with additional genes on the 3' terminus of the RNA genome compared to subgroup I ampeloviruses. PMWaV-2, along with the presence of mealybug vectors, have been shown to be necessary factors in symptom development in Hawaii. Some of these extra genes in the 3' of PMWaV-2 have recently been shown to function as silencing suppressors, and may play a role in the virulence of PMWaV-2 and symptom development. In other regions of the world, reports of symptomatic plants without PMWaV-2 infection, but with PMWaV-1, -3 or some combination, contradict the requirement of PMWaV-2 for symptom development in MWP. It is possible that further, uncharacterized PMWaVs may be present in symptomatic pineapple plants that test negative for PMWaV-2, explaining the inconsistency in symptom development. More research is necessary to explore the confusing etiology of the MWP disease, and to perhaps shed light upon the symptom development.

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Section: Detection Of Pmwavsmentioning

confidence: 99%

Section: Pmwav-1 and -3mentioning

confidence: 99%

Mealybug Wilt of Pineapple and Associated Viruses

et al. 2018

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“…The products of PCR in this assay could then be readily detected by melting curve analysis using SYTO9 [34,35]. To date, few studies have reported nested-PCR performed in a single tube [36][37][38]. Unlike those described single-tube nested RT-PCR assays using either fluorescent probes [36], gel electrophoresis [37] or spatial separation of the outer and inner primer sets [38], the RTN RT-PCR assay described in this study combines the reaction and detection steps in a single closed tube, which can effectively prevent contamination and eliminates the needs for post-PCR electrophoresis or a fluorescent probe.…”

Section: Discussionmentioning

confidence: 99%

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease

Niu

et al. 2016

Arch Virol

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Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

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“…PCR products could be readily detected by melting curve analysis using SYTO9 (Gudnason et al, 2007;Monis et al, 2005). Some studies have reported performing nested PCR in a single tube using fluorescent probes, gel electrophoresis, or spatial separation of outer and inner primer sets (Brisco et al, 2011;Dey et al, 2012;Hu and Arsov, 2014;Moser et al, 2012). However, our OTNRT-PCR assay can effectively prevent contamination and eliminate the need for post-PCR electrophoresis or the use of a fluorescent probe, and complete detection takes only 3 h. Furthermore, the running costs of the OTNRT-PCR assay are 10% less than the TaqMan probe qRT-PCR assay.…”

Section: Assaymentioning

confidence: 99%

A highly sensitive 1-tube nested real-time RT-PCR assay using LNA-modified primers for detection of respiratory syncytial virus

Zhao

et al. 2019

Diagnostic Microbiology and Infectious Disease

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Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid--based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02 × 10 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.

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A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2)

Cited by 9 publications

References 15 publications

Mealybug Wilt of Pineapple and Associated Viruses

Mealybug Wilt of Pineapple and Associated Viruses

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease

A highly sensitive 1-tube nested real-time RT-PCR assay using LNA-modified primers for detection of respiratory syncytial virus

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