MultNAT HIV-1 Viral Load Assay: a simple, rapid and sensitive cross-primer amplification assay for detecting the HIV-1 virus in the field

Study design

We prospectively collected and tested 366 clinical blood samples from Xixi Hospital in Hangzhou, of which 183 were negative and 183 were positive. HIV-positive participants were enrolled if their viral load exceeded 100 copies/mL per the DaAn reference test, and the enrolled negative patients were from the HIV-negative hospitalized patients and HIV-positive patients with negative DaAn loads, and the healthy medical examination population included 20 people. The HIV-1 nucleic acid quantitative detection kit (PCR-fluorescent probe method) purchased from DaAn Gene Co., Ltd. is CE-IVD/NMPA-approved and widely used in clinical practice in China (Li et al., 2023). Given its high accuracy, reliability, and cost-effectiveness, we selected the DaAn Gene kit for our study. The study was approved by the Scientific Research Subcommittee of the Ethics Committee of Xixi Hospital, Hangzhou, China, with the approval number Hangzhou Xixi Medical Lun-Quan 2023 Research No. 005. Written informed consent from patients was waived, as the samples used were leftover samples from routine clinical examinations.

The MultNAT HIV-1 Viral Load Assay used in this research

The MultNAT HIV-1 Viral Load Assay (Ustar Biotechnology Co., Ltd., Hangzhou, China) includes HIV-1 automated tubes, HIV-1 RNA extracts, and HIV-1-QS quantitative internal standards. The HIV-1 automated test tube is divided into the lysis-nucleic acid binding bead zone, the magnetic bead-nucleic acid washing zone and the nucleic acid elution-nucleic acid amplification zone from top to bottom. The nucleic acid elution-nucleic acid amplification zone comprises reaction zone 1 and reaction zone 2. Reaction zone 1 is used for the detection of the LTR gene, and reaction zone 2 is used for the detection of the pol gene (Fig. 1A). The HIV-1 automated test tube must be loaded into the nucleic acid analyzer (Ustar Biotechnology Co., Ltd., Hangzhou, China) for detection (Fig. 1B).

Development of the formula for quantification of HIV-1 RNA viral load

In order to determine the titer of HIV-1 RNA, we optimized the system of internal quantitative standard nucleic acid in the HIV-1-QS which had a same amplification efficiency with the target nucleic acid. Viral load determination is based on the assumptions that the target nucleic acid and the internal quantitative standard nucleic acid are amplified with the same efficiency and that equal amounts of amplicon copies of target nucleic acid and quantitative standard nucleic acid are amplified and detected at the same cycle threshold (Ct). Therefore, the ΔCt (Ct_QS−Ct_target) is linear to log (target concentration/QS concentration), where in “QS” stands for the internal quantitative standard nucleic acid. For instance, the viral load of HIV-1 RNA can then be calculated by using a polynomial calibration formula as in the following equation: (1)${\displaystyle \text{Viral load of HIV-1 RNA (cps/mL)}=10^{\left(\mathrm{a}{\left(\Delta \mathrm{Ct}\right)}^2+\mathrm{b}\left(\Delta \mathrm{Ct}\right)+\mathrm{c}\right)}}$ (2)${\displaystyle \Delta \mathrm{Ct}={\mathrm{Ct}}_{\mathrm{QS}}-{\mathrm{Ct}}_{\mathrm{target}}.}$

According to the ΔCt Eq. (2) of standard AcroMetrix™ HIV-1 Panel and HIV-1-QS, we determined the polynomial constants (a, b and c) in the Eq. (1). For a specific target, the polynomial constants (a, b and c) and the concentration of the quantitative standard nucleic acid are known, therefore the only variable in the equation is the difference ΔCt. The polynomial constants for the LTR target were a =  − 0.0014, b = 0.2248, c = 4.2102, and for the pol target were a = 0.0195, b = 0.3969, c = 3.4798. The fit formula had been built in the MultNAT instrument and the viral load of HIV-1 RNA can be interpreted automatically.

Primers and probes used in the MultNAT HIV-1 Viral Load Assay

The MultNAT HIV-1 Viral Load Assay was designed with primer pairs and probes for the HIV-1 LTR and pol genes, and the primers were synthesized by Shanghai Sangong Biotechnology Co. (Shanghai, China) (Table 1 & Fig. 2). To assay the samples, extracts were chemically lysed at 94 °C in a nucleic acid analyzer (Ustar Biotechnology Co., Ltd., Hangzhou, China) to release nucleic acids. The nucleic acids from the samples that were used for the assay were permeated through the nucleic acid magnetic guide of the nucleic acid analyzer, passed through the different detection zones, purified in the magnetic bead–nucleic acid wash zone, and finally eluted at the bottom of the nucleic acid detection tube, where the amplification reaction occurred. With the use of the hot-start Taq enzyme and reverse transcriptase, the target sequences were amplified under isothermal conditions via amplification primers. Moreover, the fluorescent probe specifically bound to the target and generated a fluorescent signal. The fluorescence signal could be captured in real time during the nucleic acid amplification process and analyzed to automatically determine the test result and calculate the amount of HIV-1 RNA in the sample (in copies/mL) according to the built-in quantitative formula. The developed kit is a fully automated method for nucleic acid analysis, i.e., lysis, binding, washing, elution, and amplification reactions are performed in a closed assay tube.

Generation of a standard RNA template

The RNA template of HIV-1 LTR gene is from the HIV-1 RNA standard quantitative synthetic human immunodeficiency virus 1 (HIV-1) RNA (Cat: VR-3245SD) which is purchased from ATCC; the HIV-1 pol gene RNA template was transcribed from the pol plasmid template, the pol plasmid template was synthesized by Sangon Biotech (Shanghai, China) and the transcription reagents were purchased from Nanjing Novozymes Bioscience Co. (Nanjing, China).

Sensitivity and specificity assessment of the MultNAT HIV-1 viral load assay

We used the AcroMetrix™ HIV-1 Panel from Thermo Fisher Scientific (Waltham, MA, USA) to assess the sensitivity of the Human Immunodeficiency Virus Type 1 Nucleic Acid Load Assay, and the concentration and quality of the extracted DNA were assessed via the MultNAT Molecular Diagnostic PCR System (Ustar Biotechnology Co., Ltd., Hangzhou, China). The concentration and quality of the extracted DNA were assessed. MS2 phage broth (BeNa Culture Collection) was used as the negative serum substrate, and the AcroMetrix™ HIV-1 Panel was diluted with the negative serum substrate to obtain test concentrations of 1,000, 500, 100, and 50 copies/mL. Then, these samples were added to assay tubes and assayed on a fully automated medical PCR analyzer system with three replicates for each concentration. The lowest diluted concentration that was positive in all three replicates was defined as the limit of detection.

To further determine the specificity of the system, we utilized this kit to validate common clinical pathogens (human immunodeficiency virus type 2, human T-cellophilic viruses 1 and 2, Candida albicans, cytomegalovirus, human herpesvirus 4, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus 1 and 2, human herpesvirus 6, influenza A virus, Staphylococcus aureus, etc.). The test was repeated three times for each common clinical pathogen.

Clinical application of the MultNAT HIV-1 Viral Load Assay

From June to August 2023, HIV-negative and HIV-positive patients who underwent medical checkups at Xixi Hospital in Hangzhou City, Zhejiang Province, China, were prospectively enrolled in this study. The blood samples collected in this study were provided by the Laboratory Department of Xixi Hospital in Hangzhou City, China. The obtained blood samples were centrifuged at 3,000 rpm to obtain serum, which was evenly divided into two portions.

Statistical analyses

The differences between the detection rates of the two methods were tested via McNemar’s test. A P value of < 0.05 was considered statistically significant.

Sensitivity of the MultNAT HIV-1 viral load assay

We diluted the purchased AcroMetrix™ HIV-1 Panel to the appropriate concentration and measured the sensitivity of the Human Immunodeficiency Virus Type 1 Viral Load Assay for detecting HIV-1 RNA to 100 copies/ml (Table 2).

Specificity of the MultNAT HIV-1 Viral Load Assay Kit

Our analysis of the 14 common clinical pathogens revealed no positive results, indicating that the MultNAT HIV-1 Viral Load Assay Kit (Ustar Biotechnology Co., Ltd., Hangzhou, China) has no cross-reactivity with the 14 common clinical pathogens (Table 3).

Clinical performance of the MultNAT HIV-1 Viral Load Assay Kit

A total of 366 clinical samples were tested, 183 of them were negative, and 183 were positive based on the DaAn results (Table 4). Among 366 samples, only one patient with confirmed positive HIV viral load was diagnosed negative by MultNAT HIV-1 Viral Load Assay, but all the others were fully matched.

The accuracy of the MultNAT Molecular Diagnostic PCR System and its consistency with clinical diagnosis were as follows: kappa value of 0.9945; positive compliance rate of 99.45% (95% CI [98.03%–99.93%]); negative compliance rate of 100% (95% CI [97.99%–100.00%]); and total compliance rate of 99.73% (95% CI [97.99%–100.00%]). The positive compliance rate, negative compliance rate and total compliance rate were all ≥95%. The linear correlation coefficient (r) between the kit used in the present study and the comparison kit was 0.95, the R2 was 0.90, and the correlation coefficient was y = 0.989x−0.0322 (Table 4 & Fig. 3), which proved that the correlation coefficient between the kit used in the present study and the comparison kit was good.