US 2004/0072148 A1 · Simultaneous detection of HBV, HCV, and HIV in plasma samples using a multiplex capture assay

Abstract

The present invention is directed to a capture assay to simultaneously screen for HBV, HCV and HIV nucleic acids in samples such as plasma. The nucleic acids including both viral DNA and RNA are purified from the plasma samples in a single extraction procedure. In one embodiment, a mixture of degenerate biotin-labelled PCR primers specific for the HBV, HCV, HIV-1 type M and HIV-1 type O are used to amplify any of these viruses which may be present in plasma. Amplified products are captured by hybridization to immobilized capture sequence, and thereafter detected. An internal control vector containing a synthetic fragment flanked by sequences corresponding to the HBV primers was designed to monitor sample recovery during extraction, amplification and detection. All major subtypes of HBV, HCV and HIV including HIV-1 type O have been confirmed and detected by the assay.

Claims46

Dependent claims

A method for detecting the presence of multiple viral agents in a test sample, comprising:

a. carrying out an amplification reaction amplifying nucleic acids from at least one or more of HIV, HCV and HBV using a mixture of primers specific for HBV, HCV, HIV-1 type M and HIV-1 type O;
and
b. detecting for the presence of amplified nucleic acids and determining whether said nucleic acids are associated with at least HIV, HCV, or combinations thereof.

A kit comprising one or more vials containing a primer specific to HIV, HBV, HCV or combinations thereof;

wherein said primers specific to HIV are selected from the group consisting of polynucleotides of 10 to 100 bases capable of hybridizing under stringent conditions to a nucleic acid having a sequence selected from the group consisting of
a 5′ ATACCCATGTT(C/T)(A/T)CAGCATTATCAGA3′;
b. 5′ CTATTTGTTC(C/T)TGAAGGGTACTAGTA 3′;
C. 5′(G/T)AATTTGCTCTTGCTG(G/T)GTGCTAGTT 3′;
d. 5′ ATTCCTATGTT(C/T)ATGGCATT(G/A)TCAGA 3′;
or
e. a complement of any one of the sequences listed in a to d;
wherein said primers specific to HBV are selected from the group consisting of
f. polynucleotides capable of hybridizing under stringent conditions to a sequence of HBV strain 1366h pre-S2-S protein (S) gene (GenBank accession number (A#) AF214659) or the complement thereof, wherein said sequence is from nucleotide 300 to nucleotide 400, or a portion thereof comprising at least the sequence 340 to 350;
and
g. polynucleotides capable of hybridizing under stringent conditions to a sequence of HBV strain 1366h pre-S2-S protein (S) gene (GenBank accession number (A#) AF214659) or the complement thereof, wherein said sequence is from nucleotide 650 to nucleotide 750, or a portion thereof comprising at least the sequence 710 to 720;
and
wherein said primers specific to HCV are selected from the group consisting of
h. polynucleotides capable of hybridizing under stringent conditions to a sequence of HCV polyprotein gene (GenBank accession number (A#) AF271632) or the complement thereof, wherein said sequence is from nucleotide 50 to nucleotide 150, or a portion thereof comprising at least the sequence 83 to 93;
and
i. polynucleotides capable of hybridizing under stringent conditions to a sequence of HCV strain MD12 complete genome (GenBank accession number (A#) AF207753) or the complement thereof, wherein said sequence is from nucleotide 220 to nucleotide 320, or a portion thereof comprising at least the sequence 271 to 281.

13 more elements

Dependencies are inferred from the claim text.

Simultaneous detection of HBV, HCV, and HIV in plasma samples using a multiplex capture assay

Life of this patent

Simultaneous detection of HBV, HCV, and HIV in plasma samples using a multiplex capture assay

Life of this patent

Abstract

Claims46