Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1

Abstract

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5' terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4 degrees C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA.

FIG. 1.

Method for construction of the exogenous chimeric fragment pol-3034b. During the first-round PCR, two parts (fragments A and B; nucleotides 2125 to 3310, with 1,186 bp, and nucleotides 3311 to 5101, with 1,791 bp, respectively) of the nearly entire HIV pol sequence were amplified from the template plasmid pSG3▴env (kindly provided by the National Center for AIDS/STD Control and Prevention, China CDC; containing the entire HIV RNA genome; GenBank accession no. AB221005) using primers 1 and 2 and primers 3 and 4, respectively. In the second-round PCR, fragment C was obtained from fragment B by using primers 5 and 4 to prepare for overlapping extension PCR. Then the 3,034-base exogenous chimeric sequence (pol-3034b) was obtained by overlapping extension PCR using primers 1 and 4 from templates (fragments A and C).

FIG. 2.

Results of electrophoresis of AR-HIV-pol-3034b after purification by gel exclusion chromatography (1% agarose gel). Lane M, DNA marker; lane 1, AR-HIV-pol-3034b.

FIG. 3.

Results of RT-PCR and PCR with 1% agarose gel analysis. Lane 1, RT-PCR of RNA extracted from AR-HIV-pol-3034b; lane 2, pSG3▴env, the positive control; lanes 3 to 6, the four negative controls (H₂O, H₂O after extraction and RT, RNA extracted from VLPs without RT, and VLPs without extraction and RT).

FIG. 4.

Resistance of purified AR-HIV-pol-3034b particles to nucleases. Lanes 1, 3, and 5, AR-HIV-pol-3034b, pACYC-MS2-pol-3034b, and RNA isolated from AR-HIV-pol-3034b, respectively, before incubation in RNase A and DNase I. Lanes 2, 4, and 6, AR-HIV-pol-3034b, pACYC-MS2-pol-3034b, and RNA isolated from AR-HIV-pol-3034b, respectively, after incubation in RNase A and DNase I at 37°C for 60 min. AR-HIV-pol-3034b was completely resistant to DNase and RNase treatment; however, naked DNA and RNA were both degraded rapidly.

FIG. 5.

Study of the stability of AR-HIV-pol-3034b. AR-HIV-pol-3034b samples of different concentrations in normal human EDTA-preserved plasma were stable at 4°C for at least 6 months. The key identifies high- and low-copy-number samples by their concentrations (150,000 and 500 copies/ml, respectively).

FIG. 6.

Comparison of the AR-HIV-pol-3034b positive controls and the Versant HIV-1 RNA 3.0 assay controls in a clinical setting. The CVs for the AR-HIV-pol-3034b positive controls and the Versant HIV-1 RNA 3.0 assay controls were comparable. The high- and low-positive AR-HIV-pol-3034b controls both performed reliably compared with the corresponding controls provided with the kit.

FIG. 7.

Linearity of AR-HIV-pol-3034b in the Versant HIV-1 RNA 3.0 assay. AR-HIV-pol-3034b samples were diluted in serial 10-fold dilutions throughout the range of the HIV bDNA test. Tenfold dilutions of AR-HIV-pol-3034b produced linear results (y = 0.14 + 0.97x; r² = 0.997). The three differently colored symbols represent three observed concentrations for each expected concentration.

FIG. 8.

Correlation of the AR-HIV-pol-3034b standards with the commercial RNA standards provided with the kit, determined by processing 46 clinical samples with each set of standards separately. The relationship between the standards is defined by the equation y = 15.40 + 1.05x (r² = 0.984).