KRISHI: Technology Repository

General Part-1

Technology Code:-	:	201596858305078
Organization Details...
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Details of Inventors..
Principal Inventor	:	Dr. G. Venkatesh
Principal Inventor Designation:	:	Principal Scientist
Principal Inventor Email	:	G.Venkatesh@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. D. Senthil Kumar,Dr. S. Nagarajan,Dr. Richa Sood
Co-Inventor Email	:	Senthil.D@icar.gov.in,S.Nagarajan@icar.gov.in,Richa.Sood@icar.gov.in
Technology Name	:	Lateral flow test for rapid detection of H5 avian influenza virus antigen in poultry
Technology Details..
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Complete Details of Technology:	:
India has been suffering from continual outbreaks of H5N1 highly pathogenic Avian Influenza Virus (HPAIV) since 2006 and these outbreaks had resulted in great economic loss to the huge poultry industry and have raised the pandemic threat. Even though various laboratory based virological and molecular biological tests are available for the diagnosis, it is necessary to concentrate on preliminary screening and diagnosis at the farm level, as it facilitates the decision-making process and speeds up the implementation of control measures. The lateral flow test (LFT) is a novel immuno-chromatography based immunoassay which provides an economic, simple and rapid approach, which is especially suitable for a wide variety of field applications without the use of instruments. The test employs a immuno-chromatography test (ICT) strip coated with two different antibodies in the membrane in the form of two lines and a pad impregnated with conjugated detector antibody. Monoclonal antibodies (MAb) were developed against H5N1 and a panel of affinity purified MAbs, and H5-polyclonal antibody against the recombinant H5HA1 portion were evaluated for their potential to be used as a pair of detector and capture antibodies. The antibodies were conjugated with gold nanoparticles and used in a checker board format for evaluation. A pair of Mabs were selected for development of test. The test was further optimised considering various variables such as gold conjugation parameters, buffer conditions for capture antibody, membrane size and sample size etc.,. the The test strip has an upper line which is the control line and the bottom line which is the test line coated with a H5 specific monoclonal antibody. If the sample is positive, both control and test line of reddish purple colour will appear. If the sample is negative, only the upper control line will be visible. If only the lower test line appears or no lines appear, the test shall be considered invalid. To determine its analytical sensitivity and specificity, the strips were tested with reference antigen from Influenza A virus (IAV) subtype 1 to 16. The strips were also tested with 43 isolates of AIV covering four clades of AIV H5N1 (2.2, 2.2.1, 2.3.2.1a and 2.3.2.1c) to test for its inclusivity and seven subtypes of IAV (H1N1, H3N8, H4N6, H6N2, H7N9, H9N2, H11N9) and one Newcastle disease virus available in the lab to test for its exclusivity. The isolates were from 12 States/UT - Maharashtra, Orissa, Kerala, Bengal, Assam, Manipur, Tripura, Gujarat, UP, Bihar, Chattisgarh, Chandigarh. The strips reacted with all the tested clades of H5 AIV viruses only. One virus each of clade 2.2 and 2.3.2.1a with known HA titres were used for assessing analytical sensitivity. The viruses were diluted serially two fold and the dilutions were spiked in replicates of cloacal and oro-pharyngeal swabs. Twenty each of oro-pharyngeal swabs and cloacal swabs collected from SPF chicken were spiked with 20 H5 viruses of various clades (2.2, 2.2.1, 2.3.2.1a and 2.3.2.1c) with varied HA titres. The strips could detect all the viruses. The 252 samples from three groups of chicken infected with 3 different clades of viruses were collected and used for evaluation of diagnostic sensitivity and specificity. The test showed a sensitivity of 53.7% and specificity of 100%. The prepared strips were stable at room temperature for 6 months tested so far. The test kit was prepared with ICT strips, disposable Swabs, Sample buffer in tubes, disposable droppers, assay tubes and kit information sheet. The intra-lab and inter-lab validation of the kit was done using a chicken cloacal and oro-pharyngeal swabs spiked with a panel of viruses . The test was validated by three working groups in the institute viz., Vaccine and diagnostics group, Avian diseases group and Ruminant diseases group. The test was validated by four external institutes/ labs viz., a). CADRAD, ICAR-IVRI, Izatnagar. b). ICAR-NRCE, Hisar. c). SRDDL, IAH&VB, Bengaluru d). NIV, Pune.
Brief Description of Technology Including Salient Features:
The Lateral flow test for rapid detection of H5 Avian Influenza Virus antigen in poultry was named ICAR - AIV H5 Antigen Lateral Flow Test (IA5LFT) is an ICT based rapid immunoassay. The test is intended for rapid qualitative detection of Avian Influenza virus (AIV) subtype H5 antigen in tracheal and cloacal swab samples from H5N1 AIV infected chicken. The kit contains ICT strips, swabs, sample buffer in tubes and assay tubes. The ICT strip is coated with two different indigenously developed monoclonal antibodies in the membrane in the form of two lines. The upper line is the control line and the bottom line is test line coated with a H5 specific monoclonal antibody. If the sample is positive, both control and test line of reddish purple colour will appear. If the sample is negative, only the upper control line will be visible. If only the lower test line appears or no lines appear, the test shall be considered invalid. Positive samples should be submitted to a reference lab for confirmation
Benefits/Utility	:
The test is easy to perform and interpret at the field level. This test kit is indigenously developed and would be an import substitute for rapid field diagnosis of H5N1 AIV infections in poultry. The test can be carried out at the doorstep of the farmer without requirement of any instruments. The kit can be used for routine surveillance for detection of H5 antigen in H5N1 AIV infected chicken at the field level. This would enable earlier preparation for control measures.
Precaution With The Technology	:	The kit is to be stored between 4 �C to 28 �C and not to be frozen. The ICT strips are provided in airtight container with desiccant. Perform the test immediately after removing the strip from the container as it is sensitive to humidity and heat.
How To Use	:
1. Collect the tracheal/ oropharyngeal swabs/ cloacal swabs from dead/sick birds. 2. Place the tube with sample buffer in a stand. Immerse the swab in the sample buffer and rotate the swabs for 15 � 30 seconds. Then discard the swab after squeezing it by pressing against the side of the tube 3. Wait for 5 minutes for settling down of the large particles. (Alternatively, can be clarified by centrifugation at 2000 rpm for 2 minutes). Take the supernatant from the tube using the dropper provided and add six to eight drops in the provided assay tubes. (Ensure that no sediment/debris is picked up). 4. Place the ICT strips in the assay vials with lower end immersed in the sample. Read the result after 20 minutes. Reading the results beyond 25 minutes is not recommended Interpretation of results a. The test is considered Positive when both control line and test line develop in the strip. The developed lines will be of reddish purple in colour b. The test is considered negative when only the control line develop c. The test is considered invalid when only the test line develop
Impact, If Adopted	:
The test kit can be used during suspected AIV outbreaks and testing of clinical samples by field veterinarians, diagnosticians, the regional, national and international laboratories involved in poultry disease diagnosis.
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	:	Lateral flow test, H5 Avian Influenza Virus, antigen detection, poultry

Technology Development Details Part-2

Project Details (Through which technology was developed)	:	Title of the project: Development of antibody based rapid test for detection of H5 Avian Influenza Virus in poultry. Funded by: ICAR-Consortia Research Platform on Vaccines & Diagnostics
Time of Initiation Technology Development	:	9-2015
Time of Completion Technology Development	:	8-2019
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)	:	National Institute of Virology, Pune
Year of Release/Adoption(YYYY)	:	1-2020

Applies To(Regional Differentiation)Inform Part-3

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