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Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

ABSTRACT: Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect???141 fg of RNA when cDNA used as a template. The assay detected???0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15-20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

SUBMITTER: Ghosh DK

PROVIDER: S-EPMC7693335 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

Ghosh Dilip Kumar DK Kokane Sunil B SB Gowda Siddarame S

Scientific reports 20201126 1

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbre ...[more]

PMID: 33244066

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Project description:Potatoes are developed vegetatively from tubers, and therefore potato virus transmission is always a possibility. The potato leafroll virus (PLRV) is a highly devastating virus of the genus Polerovirus and family Luteoviridae and is regarded as the second-most destructive virus after Potato virus Y. Multiple species of aphids are responsible for the persistent and non-propagating transmission of PLRV. Due to intrinsic tuber damage (net necrosis), the yield and quality are drastically diminished. PLRV is mostly found in phloem cells and in extremely low amounts. Therefore, we have attempted to detect PLRV in both potato tuber and leaves using a highly sensitive, reliable and cheap method of one-step reverse transcription-recombinase polymerase amplification (RT-RPA). In this study, an isothermal amplification and detection approach was used for efficient results. Out of the three tested primer sets, one efficiently amplified a 153-bp product based on the coat protein gene. In the present study, there was no cross-reactivity with other potato viruses and the optimal amplification reaction time was thirty minutes. The products of RT-RPA were amplified at a temperature between 38 and 42 °C using a simple heating block/water bath. The present developed protocol of one-step RT-RPA was reported to be highly sensitive for both leaves and tuber tissues equally in comparison to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. By using template RNA extracted employing a cellular disc paper-based extraction procedure, the method was not only simplified but it detected the virus as effectively as purified total RNA. The simplified one-step RT-RPA test was proven to be successful by detecting PLRV in 129 samples of various potato cultivars (each consisting of leaves and tubers). According to our knowledge, this is the first report of a one-step RT-RPA performed using simple RNA extracted from cellular disc paper that is equally sensitive and specific for detecting PLRV in potatoes. In terms of versatility, durability and the freedom of a highly purified RNA template, the one-step RT-RPA assay exceeds the RT-PCR assay, making it an effective alternative for the certification of planting materials, breeding for virus resistance and disease monitoring.

Project description:Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and probes targeting the 18S rRNA gene of both T. equi and B. caballi, the ema-1 gene of T. equi and the bc48 gene of B. caballi were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R2 value was 0.983 for the concentration range 0.2 ng (4.1 × 107 DNA copies) to 2.0 fg (4.1 × 101 DNA copies). For the pUC57-ema gene DNA, the R2 value was 0.993 for the concentration range 0.2 ng (5.26 × 107 DNA copies) to 2.0 fg (5.26 × 102 DNA copies). For the pUC57-Bc18S gene DNA the R2 value was 0.976 for the concentration range 2.0 ng (4.21 × 108 DNA copies) to 2.0 fg (4.21 × 102 DNA copies). For the pUC57-Te18S gene DNA, the R2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 108 DNA copies) to 2.0 fg (4.16 × 102 DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the bc48 gene of B. caballi and the 18S rRNA gene of T. equi is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing T. equi and B. caballi, showing great potential for on-site EP screening of horses.

Dataset Information

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

Publications

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

Similar Datasets

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