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KRISHI: Technology Collections Developed by ICAR Institutes

General Part-1



Technology Code:- : 201596626809418
Organization Details...
Subject Matter Division : {{smdOb.smdName}}
Organization Name : {{orgOb.orgName}} ,{{orgOb.City}}
AICRP name if any (AICRP) : All Not Applicable {{aicrpOb.aicrpName}}
Details of Inventors..
Principal Inventor : Dr. Atul Kumar Pateriya
Principal Inventor Designation: : Scientist
Principal Inventor Email : Atul.Pateriya@icar.gov.in
Principal Inventor Address : ICAR-National Institute of High Security Animal Diseases, Anand Nagar, Hathai Kheda Road, Bhopal - 462022 Madhya Pradesh, India
Co-Inventor Name : Dr.S.Nagarajan, Dr.S.Bhatia, Dr.A.A.Raut, Dr.Richa Sood, Dr.Naveen Kumar, Dr.D.D.Kulkarni
Co-Inventor Email : S.Nagarajan@icar.gov.in,sandeep.bhatia1@icar.gov.in,Ashwin.Raut@icar.gov.in,Richa.Sood@icar.gov.in,Naveen.Kumar4@icar.gov.in,ddkulkar@gmail.com
Technology Name : Multiplex Real Time RT-PCR Kit for Avian Influenza A virus Typing and H5 and H9 Subtyping
Technology Details..
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Complete Details of Technology: :
Avian influenza (AI), commonly known as bird flu has been responsible for huge economic losses to the poultry farmers in India through several outbreaks and culling of chickens. The laboratory testing of clinical samples from poultry and other avian species during emergency (high mortality and morbidity) is done by virological methods (agent identification) including the isolation of virus and genome detection by molecular testing. In emergency situations, the samples are tested primarily by RT-qPCR assay due to its higher sensitivity and specificity and production of results within few hours (4-6 hrs). On the basis of these results further action is taken to control the disease by implementation of control program. The implementation of control program also depends upon the identification of notifiable subtypes (H5/H7) of influenza virus. The infection of AI H5 subtype causes heavy mortality in poultry (upto 100%); also AI H9 subtype, which is prevalent and frequently isolated in Indian poultry, also causes high morbidity and mortality in co-infection with other viral (NDV) and bacterial diseases. Therefore it becomes very important to identify and differentiate both H5&/H9 subtypes of influenza infection to implement specific control measures in field. Since RT-qPCR assays are conducted in a fixed format of 96 well qPCR block including control reactions (positive, negative and no template control); therefore, only limited RNA samples can be tested in singlex format of taqman RT-qPCR for both Influenza A typing and subtyping (differentiation of H5 and H9). Testing of each sample individually for all the 03 targets (Influenza A typing, H5 and H9 subtyping) also add cost and labor towards the testing. Therefore, as part of high throughput testing tool, realtime multiplex Taqman RT-qPCR (MP RT-qPCR) kit has been developed for simultaneous influenza A typing and H5 &/or H9 subtyping using uniform reaction conditions. This kit will definitely reduce the cost of testing and enhance the diagnostic/testing capacity by three times. This kit can be used for testing of routine (surveillance) and clinical samples (outbreak/emergency situation) as screening and confirmatory test. The kit has been optimized and validated on oro-pharyngeal and naso-pharyngeal swab, cloacal swabs (and faecal material), and tissues of poultry, duck and other domestic and wild birds. Details of Kit component and test run criteria: The kit consists of PCR mix, primer-probe mix (in-house designed and validated), RT enzyme and positive controls. The user has to mix all the components in a recommended volume followed by addition of test RNA sample from oral/nasal swab, cloacal swabs (and faecal material), and tissues of poultry, duck and other domestic and wild birds. After running the qPCR as per the recommended thermal profile, user can easily diagnose the influenza type A and H5-H9 infection by analyzing the results against three different reporter dyes namely FAM (495-520nm) for M probe, HEX (535-556nm) for H5 probe and Cy5 (675-954nm) for H9 probe. Analytical and Diagnostic performance of the kit: The kit has been validated as per the OIE criteria. On evaluation of analytical performance of the kit, M probe (Type A) detected RNA from H1 to H16 influenza subtypes (inclusivity); whereas H5 and H9 probes respectively detected H5 and H9 influenza subtype only (exclusivity). The kit is able to detect upto 1 EID50 of reference avian influenza H5N1 and H9N2 viruses or 10-100 copies of in-vitro transcribed RNA (IVT-RNA) for all the three targets. The diagnostic sensitivity (DSe) and specificity (DSp) of the assay was evaluated to 99.7% and 100% respectively.
Brief Description of Technology Including Salient Features:
The purpose of this kit is to confirm the presence of Influenza Type A infection and H5 &/or H9 subtypes of suspect or clinical cases. The RNA extracted from the test sample is subjected to multiplex realtime Taqman RT-qPCR assays with three different sets of primers and probes (M gene for influenza A typing, HA gene of H5 and H9 subtypes) in a single reaction for detection of Influenza type A infection and, differentiation of H5 and H9 subtypes. The oligos (06 primers and 03 probes) of the kit are novel, in-house designed and can be used on any qPCR-based platform compatible for multiplexing with FAM (Type A), HEX (H5) and Cy5 (H9) reporter dye) filter. Users can easily identify/diagnose Influenza type A infection and H5 and H9 subtype infection against the amplification of any of the target.
Benefits/Utility :
Poultry is one of the fastest growing segments of the agricultural sector in India. The recurrent outbreaks of Bird flu (Avian Influenza) in poultry are causing great economic losses to the poultry farmers of India. The indigenous multiplex realtime RT-qPCR kit developed shall be the replacement/alternative of traditional singlex RT-qPCR assays/kits for avian influenza diagnosis to increase the diagnostic capacity. This kit can perform three assays in a single tube reaction (Typing and H5-H9 subtyping). The use of this kit shall reduce the cost, time and manpower required for bird flu diagnosis for effective implementation of bird flu control program. The user of the kit can be national & international laboratories that provide diagnostic services for Avian Influenza.
Precaution With The Technology : The test should be conducted and interpreted by any laboratory personal that has been trained to perform qPCR. The kit literature should be followed strictly for interpretation of test results especially for borderline positive cases. The positive controls of the kit should be stored separately in -800C to avoid any cross contamination.
How To Use :
Following all the instructions and precautions, user shall arrange all the necessary plasticware and instruments/equipment’s desired from user end. All the kit components shall be handled only at the recommended temperature and facility as mentioned in the kit manual. 1. User shall prepare the “Layout Plan” as per the number of samples to be tested and accordingly calculations shall be done with extra 10% reactions to compensate the volumetric error. 2. The mastermix shall be prepared in Mastermix preparation room as per the sequence/order of dispensing given in kit manual. 3. The 19 µl mastermix shall be dispensed in each test well of plate/tube/strip for each reaction. 4. Dispensing of test RNA shall be conducted in dedicated “template dispensing room” as per the “Layout Plan”. One micro litre of nuclease free water shall be dispensed in NTC well and rest of the wells shall be dispensed with 1 µl test RNA. 5. The positive controls shall be dispensed at the end of plate set-up. 6. After giving a brief spin the qPCR run shall be started in qPCR machine as per thermal profile recommended in kit manual with selection of FAM, HEX and Cy5 reporter Dye filter. 7. The validity of Run shall be confirmed following the results of control reactions. 8. After ensuring the valid Run, user shall interpret and record the results as per kit criteria (Ct values with sigmoid amplification curve). 9. User shall prepare the report and any unusual or borderline, intermediate results shall be correlated with other tests including alternate TaqMan RTqPCR assays and/or virus isolation.
Impact, If Adopted :
All the regional, national & international laboratories authorized to provide diagnostic services for Avian Influenza can use this kit (surveillance or testing of clinical/emergency samples from field).
Social Impact :
The kit may have greater social impact if, highly pathogenic avian influenza (H5 subtype) is diagnosed in early stage of infection to implement control measure to reduce economic losses and further spread of infection to nearby poultry and associated poultry workers.
TargetUsers/Stake holders : The kit can be used by all the regional, national & international laboratories authorized to provide diagnostic services for Avian Influenza.
Technology Contact..
Name : Director
Email : director.nihsad@icar.gov.in
Phone Number : 0755-2759204
Fax Number : 0755-2758842
Address : ICAR-National Institute of High Security Animal Diseases,Anand Nagar, Bhopal-462021.,Bhopal-462021
Keyword for Technology : Multiplex Real Time Reverse Transcriptase PCR,Avian Influenza virus,A typing and differentiation of H5 and H9 subtypes


Technology Development Details Part-2

Project Details
(Through which technology was developed)
: DBT-NER-Advanced Animal Disease Diagnosis and Management Consortium (ADMaC)
Technology Validated by : Within ICAR
Technology Validation Details..
Subject Matter Division : {{smdOb.smdName}}
Organization Name(if within ICAR) : ICAR-National Institute of High Security Animal Diseases,Bhopal
Organization Name(if outside ICAR,Please enter) :
Year of Release/Adoption(YYYY) : 1-2020


Applies To(Regional Differentiation)Inform Part-3

Location...
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Commodity Details..
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