General Part-1

Technology Code:-	:	201713974524792
Organization Details...
Subject Matter Division	:	{{smdOb.smdName}}
Organization Name	:	{{orgOb.orgName}} ,{{orgOb.City}}
AICRP name if any (AICRP)	:	All Not Applicable {{aicrpOb.aicrpName}}
Division name if any	:	Crop Protection
Principal Inventor	:	A. Ishwara Bhat
Principal Inventor Designation:	:	Principal Scientist
Principal Inventor Email	:	ishwarabhat.a@icar.gov.in
Principal Inventor Address	:	Principal Scientist ICAR-Indian Institute of Spices Research Marikunnu Post Kozhikode 673012 Kerala
Technology Name	:	Development of reverse transcriptase-recombinase polymerase amplification (RT-RPA) assay for rapid detection of large cardamom chirke virus
Technology Details..
Major resource	:	All Not Applicable {{majorOb.majreName}}
Minor Subject Classification	:	All Not Applicable {{minorOb.minorName}}
Minor Subject Sub Classification	:	All Not Applicable {{minorOb.minorsubName}}
Technology Group	:	All Not Applicable {{techgroup.tecg_group_name}}
Technology Related To	:	All Not Applicable {{techrela.tr_name}}
Complete Details of Technology:	:
Large cardamom (Amomum subulatum) belongs to the family Zingiberaceae and is a perennial spice crop grown in moist shady areas along hilly slopes at an elevation of 2500-5500 ft in the north-eastern states of India, Nepal, and Bhutan. Of the two viral diseases infecting large cardamom, chirke is serious with incidence ranging from 19–35% causing yield loss of up to 85%. The disease is characterized by mosaic streaks on the leaves which gradually coalesce and eventually turn brownish and dry up. The flowering in the diseased plant is extremely reduced and only 1-5 flowers develop in one inflorescence as against 16-20 flowers borne by healthy plants. Besides vegetative means, LCCV is transmitted through sap and also by aphids, Rhopalosiphum maidis as well as Myzus persicae in a non-persistent manner. The disease is caused by the large cardamom chirke virus (LCCV) (Genus: Macluravirus; Family: Potyviridae) and is prevalent in all large cardamom growing regions. LCCV is monopartite with flexuous filamentous particles of about 625-650 nm in length and 12.2 nm wide containing a linear, single-stranded, positive sense RNA of about 8 kb in size. Currently, polyclonal antiserum-based assays such as enzyme-linked immunosorbent assay (ELISA), dot-immunobinding assay (DIBA), and lateral flow immunoassay (LFIA) have been reported for the detection of LCCV. Serological assays are less sensitive compared to nucleic acid-based assays such as PCR and real-time PCR assays. However, these assays require expensive equipment, and more time to perform the tests, thus limiting the usage of these assays for large-scale screening of plants. In contrast, isothermal assays such as recombinase polymerase amplification assay (RPA) can be performed without the need for a sophisticated laboratory in a short time. RPA is performed using two primers at a constant temperature of ~39 °C for 15-30 min using primers, recombinase, single-stranded DNA binding proteins, and strand-displacing polymerase. RNA viruses can be easily detected through reverse transcriptase RPA (RT-RPA) and the products can be visualized through gel electrophoresis. RT-RPA has been successfully used for the detection of a large number of viruses infecting different crops. In the present study, we report the development and validation of RT-RPA assay for the detection of LCCV and its comparison with RT-PCR assay. Total RNA isolated by two different methods and crude extracts isolated using five different methods as templates were assessed for their ability to detect LCCV. Of these, only the total RNA isolated by both methods gave consistent and repeatable results while all the crude extracts used as templates gave non-specific amplification. RT-RPA was up to 1000 times more sensitive than conventional RT-PCR for the detection of LCCV. The detection limit of RPA was 10 fg when recombinant plasmid was used as the template. The RT-RPA assay was validated using field samples and found suitable for large-scale screening of large cardamom plants against LCCV for the selection of virus-free plants.
Brief Description of Technology Including Salient Features:
Large cardamom chirke virus (LCCV) causing chirke disease of large cardamom is a major production constraint of this crop. Rapid and accurate detection of LCCV is important for managing the disease. In the present study an isothermal assay namely, reverse transcriptase-recombinase polymerase amplification (RT-RPA) was developed for the detection of LCCV. Total RNA isolated by two different methods and crude extracts isolated using five different methods as templates were assessed for their ability to detect LCCV. Of these, only the total RNA isolated by both methods gave consistent and repeatable results while all the crude extracts used as templates gave non-specific amplification. RT-RPA was up to 1000 times more sensitive than conventional RT-PCR for the detection of LCCV. The detection limit of RPA was 10 fg when recombinant plasmid was used as the template. The RT-RPA assay was validated using field samples and found suitable for large-scale screening of large cardamom plants against LCCV for the selection of virus-free plants.
Benefits/Utility	:
The incidence of the chirke disease ranges from 19–35% causing yield loss of up to 85%. The protocol developed by us can be used for the production of virus-free plants of large cardamom rapidly without the need for a sophisticated laboratory. Planting virus-free plants in the field ensures healthy crops without disease which contributes to an increase in productivity.
Precaution With The Technology	:	There is a need to use known positive and known negative samples during testing to ensure that tests have been done properly.
How To Use	:
The test sample of large cardamom (150 mg of leaf tissue) is ground in a pestle and mortar in 2 ml freshly prepared CTAB buffer [2% cetyltrimethylammonium bromide (CTAB), 1% polyvinyl pyrrolidone (PVP), 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA]. One ml of the homogenate was incubated at 65 ºC for 15 min followed by the addition of chloroform: isoamyl alcohol (24:1). The resulting mixture was centrifuged at 11000 g for 10 min, aqueous layer was added with an equal volume of 4 M LiCl and incubated overnight at 4 ºC. On the next day, the contents were centrifuged at 13000 g for 25 min, and the pellet was washed with 70% ethanol, air-dried, and dissolved in 50 μl of nuclease-free water was used as a template for RT-RPA assay. To set the RT-RPA reaction, the supplied freeze-dried reaction pellet of the Twist Amp® basic kit was dissolved in 29.5 µl of rehydration buffer, 2.4 µl each of forward and reverse primer, 1.5 U of RevertAid reverse transcriptase (0.5 µl), 0.25 μl of RNase inhibitor and 7.45 μl nuclease-free water. The resultant mixture was aliquoted equally (8.5 μl each) into five PCR tubes, and a 1 µl template was added to the tubes. For initiating the RT-RPA reaction, 0.5 μl of 14 mM magnesium acetate solution was added and the tube was incubated for 40 min at 39 °C with moderate mixing and spinning briefly after 4 min. The RT-RPA reaction was stopped by keeping the tube at 65 ºC for 10 min and contents were loaded and run on 2% agarose gel and visualized under the GelDoc system. The presence of a band at 175 bp is considered positive for the virus.
Impact, If Adopted	:
The technology ensures the identification of LCCV-infected and healthy plants of large cardamom. The infected plants can be removed and healthy plants can be used for further propagation to produce virus-free plants through tissue culture or vegetative method. The virus-free plants produced are then used for planting in the main field. The planting of virus-free plants in the field ensures healthy crops without disease which contributes to an increase in productivity.
Social Impact	:
The virus infection can cause a yield reduction of up to 85% depending on the severity of the disease. The protocol developed can be used to identify virus-free plants to be used as mother plants for further production of virus-free plants either through tissue culture or vegetative means. Planting virus-free plants in the field ensures healthy crops without disease which contributes to an increase in productivity.
TargetUsers/Stake holders	:	Diagnostic laboratories and tissue culture laboratories involved in the multiplication of black pepper, farmers, and breeders.
Technology Contact..
Name	:	Director
Email	:	director@spices.res.in,director.spices@icar.gov.in
Phone Number	:	0495-2730294
Fax Number	:	0495-2731187
Address	:	ICAR-Indian Institute of Spices Research,Marikunnu P.O.,Calicut-673012
Keyword for Technology	:	Reverse transcriptase-polymerase chain reaction, diagnosis, sensitivity, detection, Reverse transcriptase-recombinase polymerase amplification

Technology Development Details Part-2

Project Details (Through which technology was developed)	:	Institute project, Path. XXXI: Development of off and on-site detection techniques for major pathogens of spice crops
Time of Initiation Technology Development	:	4-2022
Time of Completion Technology Development	:	6-2023
Technology Validated by	:	Within ICAR
Technology Validation Details..
Subject Matter Division	:	{{smdOb.smdName}}
Organization Name(if within ICAR)	:	ICAR-Indian Institute of Spices Research,Calicut
Organization Name(if outside ICAR,Please enter)	:
Year of Validation(YYYY)	:	10-2023
Year of Release/Adoption(YYYY)	:	3-2024
Country	:	India

Applies To(Regional Differentiation)Inform Part-3

Location...
Zone(As per the planning commission)	:	All Not Applicable {{zone.planningzoneName}}
Sub zone(As per the planning commission)	:	All Not Applicable {{zonesub.agroName}}, {{zonesub.Region}}
AgroEcological Zone(NBSS & LUP)	:	All Not Applicable {{agrozone.nbssaerName}}
AgroEcological Sub Zone(NBSS & LUP)	:	All Not Applicable {{agrosubzone.nbssaesrName}}
State Name	:	All Not Applicable {{state.stateName}}
District Name	:	All Not Applicable {{dist.distName}}
Farmer Land Holding Size	:	'All'
Farmer Type	:	'All'
Soil Type/Resource Type..
Soil Order	:	All Not Applicable {{soilorder.soilorderName}}
Soil Sub Order	:	All Not Applicable {{soilsuborder.soilsubName}}
Soil great group	:	All Not Applicable {{soilgreat.soilgreatName}}
Soil great sub group	:	All Not Applicable {{soilgreatsub.soilgreatsubName}}
Commodity Details..
Commodity	:	All Not Applicable {{commodity.commodityName}}
Commodity Type	:	All Not Applicable {{commoditytype.commoditytypeName}}
Commodity Name	:	All Not Applicable {{commodityname.commodityName}}

Publication Related To Technology Part-4

Publication Related To Technology Part-4

Research Paper information..

1. Malavika, P., Bhat, A.I., and Greeshma and M (2024 ). Development of reverse transcriptase-recombinase polymerase amplification (RT-RPA) assay for rapid detection of large cardamom chirke virus. , Virus Diseaase , 35., 1., Springer.