Simultaneous visual detection of multiple viral amplicons by dipstick assay

Abstract

A sensitive, simple, and instrument-independent method for the visual detection and identification of multiple nucleic acid amplicons by dipstick has been developed. This method is based on nucleic acid hybridization on the dipstick membrane and a signal amplification system to allow visual detection. With hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) as model analytes, it is demonstrated that the visual dipstick test combined with multiplex reverse transcription (RT)-PCR for the amplification of viral nucleic acid provides a specific and sensitive detection method. The RT-PCR products were detected by the dipstick with an efficiency similar to that of a complex, expensive, and instrument-dependent method based on fluorogenic oligonucleotide probes. The detection limits of the dipstick combined with multiplex RT-PCR were 50, 125, and 500 IU/ml for HBV DNA, HCV RNA, and HIV-1 RNA, respectively. The dipstick assay detected with similar efficiencies amplicons derived from strains of HBV genotypes A through F, HCV genotypes 1 to 6, and HIV-1 subtypes A through H as well as CRF02 circulating recombinant forms of HIV-1. Analysis of 295 clinical samples and 19 pools of 10 plasma specimens from blood donors revealed that multiplex dipstick detection was reproducible, sensitive, and specific. The visual dipstick detection of multiple amplicons thus provides an attractive alternative to complex, instrument-dependent detection methods currently in use for nucleic acid testing. This new and sensitive method for nucleic acid detection should increase the availability of genomic screening in resource-limited settings and its applicability to near-patient testing.

FIG. 1.

Design of the nucleic acid-based dipstick test. Detection of the amplification products of viral nucleic acid is achieved with two target-specific oligonucleotide probes, one for capture and one for detection. The detector probe is labeled with multiple hapten moieties and forms large lattices by specifically binding to multiple colored particle-conjugated antibodies to hapten.

FIG. 2.

Triplex dipstick detection of individual and multiple virus amplicons. Nucleic acid standards for HBV, HCV, and HIV-1 were individually amplified by RT-PCR and then tested with the dipstick to determine its ability to identify individual viruses. In addition, a mixture of plasma specimens from three individuals, each infected with one of the three viruses, was used to test the ability of the triplex dipstick to detect HBV DNA, HCV RNA, and HIV-1 RNA in the same sample simultaneously. The viral loads of the combined sample are indicated. A negative plasma sample (Neg) was used to test for the specificity of triplex dipstick detection.

FIG. 3.

Triplex dipstick detection of clinical specimens from African individuals coinfected with HIV-1, HCV, and HBV (sample identification [ID] 32), with HIV-1 and HCV (sample ID 04008), or with HIV-1 and HBV (sample ID KWADO).