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MULTIPLEX REAL TIME RT-PCR KIT FOR AVIAN INFLUENZA A VIRUS TYPING AND H5N1 SUBTYPING
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.
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.
Benifits/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.
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.
Performance:
Advantage Over Existing Process/Product
Advantage Over Existing Process/Product
Impact
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).
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.
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).
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.
Cost-Benefit Ratio
Name and Address of Firm/entrepreneur to whom the Technology has been Transferred
Special facilities Required:
Precaution with 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.
Precaution with 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.
Contact Details
Director,
ICAR-National Institute of High Security Animal Diseases,Anand Nagar, Bhopal-462021.,Bhopal-462021
Phone No.:0755-2759204, Fax No.:0755-2758842, E-mail:director.nihsad@icar.gov.in
Director,
ICAR-National Institute of High Security Animal Diseases,Anand Nagar, Bhopal-462021.,Bhopal-462021
Phone No.:0755-2759204, Fax No.:0755-2758842, E-mail:director.nihsad@icar.gov.in
ICAR Data Use Licence
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Indian Council of Agricultural Research
Krishi Bhavan, Dr. Rajendra Prasad Road, New Delhi-110 001. INDIA
Disclaimer
© 2018 All Rights Reserved •
Indian Council of Agricultural Research
Krishi Bhavan, Dr. Rajendra Prasad Road, New Delhi-110 001. INDIA