A METHOD FOR THE PREPARATION OF A KIT FOR DETECTING HEPATITIS B VIRUS (HBV) NUCLEIC ACID IN BIOLOGICAL SAMPLES

摘要

A method for preparation of a kit for capturing amplimers of Hepatitis B Virus (HBV) genome using a sense primer consisting of the sequence 5-atactgcggaactcctagc-3 [SEQ.ID. NO.l] and a anti-sense primer consisting the sequence 5- gttcacggtggtctccatgcgacgtgc-3 [ SEQ.ID. No. 2] with oligonucleotide probe having a sequence 5'-gggcgcacctctttacgcgg-3' [ SEQ. ID. NO. 3] comprising steps of i) designing oligonucleotide probe capable of capturing amplimers of HBV genome and labeled at 5' end; ii) adding biotinylated oligonucleotide probe about lµl to about 10 µl dilution buffer about 50µI to about about 100µl; iii) immobilizing the probe on a solid medium such as microwell plate, coated with streptavidin by incubating at temperature of about 37°C for about 30 minutes to about 60 minutes and washing off excess probe with wash buffer; iv) adding hybridization buffer comprising of sodium phosphate, sodium thiocyanate and Denhardts solution and incubating for period of about 15 minutes to about 30 minutes at temperature of about 37° C; v) adding amplimers of HBV genome preferably in an amount about 25µl in denatured form and incubating for period of about 30 minutes to about 60 minutes at temperature of about 42° C and washing off excess of unhybridized product by washing preferably about 5 times with wash buffer; vi) adding an enzyme conjugate preferably anti-fluorescein and incubating for the period of about 30 minutes to about 45 minutes at temperature of about 37° C and washing with wash buffer; 311/ MUM/ vii) adding substrate capable of changing colour in presence of enzyme conjugate preferably in an amount about 100 µl in absence of light, incubating at an ambient temperature for the period of about 15 minutes to about 30 minutes and adding stop solution; viii) measuring the colour developed with a suitable instrument at an appropriate wavelength preferably at 403 nm.