KRISHI: Technology Repository

General Part-1

Technology Code:-	:	201596857548890
Organization Details...
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Organization Name	:	{{orgOb.orgName}} ,{{orgOb.City}}
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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 H5N1 subtyping
Technology Details..
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Complete Details of Technology:	:
Since 2006 onwards Indian poultry is consistently facing the outbreaks of highly pathogenic avian influenza (HPAI) every year. These outbreaks has caused huge economic losses to the poultry farmers and Indian economy due to mass culling of poultry. The early detection of such outbreaks helps to prevent the further spread of disease to adjoining areas and effective implementation of avian influenza control program. The investigation of notifiable HPAI (H5/H7 subtypes) outbreak is done by laboratory diagnosis by agent identification. The clinical samples from poultry and other avian species during emergency (high mortality and morbidity) are tested by virus isolation (VI) and genome detection tests. The samples are tested primarily by taqman real-time reverse transcriptase polymerase chain reaction (RT-qPCR) tests due to its higher sensitivity, specificity and rapipdity (4-6 hrs). On the basis of these results further action is taken on field by implementation of disease control program. Since 2006 to present, outbreaks of notifiable HPAI have been reported by H5N1 subtypes only except the outbreaks of HPAI H5N8 subtype in 2016 (zoo birds). Therefore, identification of �N� subtype is also important to confirm the incursion of new subtypes either by genetic recombination (swapping or exchange of internal genes) or new introduction of virus through migratory birds etc. To ascertain the notifiable HPAI outbreak, individual RT-qPCR tests are performed in laboratory to diagnose type and subtype (H1- H16 and N1-N9) of influenza infection. These RT-qPCR tests are conducted in a fixed format of 96 well qPCR block with control reactions (positive, negative and no template control); therefore, only limited samples can be tested in singlex format of RT-qPCR for both Influenza A typing and H5-N1 subtyping. Testing of each sample individually for all the 03 targets (Influenza A typing, H5 and N1 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-N1subtyping 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-N1 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 N1 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 N1 probes respectively detected H5 among H1 to H16 and N1 among N1-N9 influenza subtype only (exclusivity). The kit is able to detect upto 1 EID50 of reference avian influenza H5N1 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 98.4% and 99% 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 & N1 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 and NA gene respectively for H5 and N1 subtypes) in a single reaction for detection of Influenza type A infection and H5 and N1 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 (N1) reporter dye) filter. Users can easily identify/diagnose Influenza type A infection and H5 and N1 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-N1 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 �l 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 �l 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 (H5N1 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 Kit, Avian Influenza, typing, H5 and N1 subtyping

Technology Development Details Part-2

Project Details (Through which technology was developed)	:	DBT-NER-Advanced Animal Disease Diagnosis and Management Consortium (ADMaC)
Time of Initiation Technology Development	:	4-2014
Time of Completion Technology Development	:	3-2019
Technology Validated by	:	OutSide 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)	:	Assam Agricultural University, Guwahati
Year of Release/Adoption(YYYY)	:	1-2020

Applies To(Regional Differentiation)Inform Part-3

Location...
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AgroEcological Sub Zone(NBSS & LUP)	:	All Not Applicable {{agrosubzone.nbssaesrName}}
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Commodity Details..
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