Xiehe Yixue Zazhi (May 2022)

Evaluation of an Assay of Zika Virus Detection Without RNA Extraction

  • LI Aqian,
  • KONG Lingjun,
  • LIU Yang,
  • YI Jie,
  • LI Chuan,
  • LIANG Mifang,
  • DOU Yaling

DOI
https://doi.org/10.12290/xhyxzz.2021-0357
Journal volume & issue
Vol. 13, no. 3
pp. 455 – 460

Abstract

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Objective To evaluate a Zika virus(ZIKV) test based on real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) that can be performed without RNA isolation. Methods The detection method is based on rRT-PCR technology, and a virus-enrichment method is used for sample processing. The sample processing procedure is: Add 112-224 μL sample treatment solution Ⅰ and 5-10 μL sample treatment solutionⅡ to 100-200 μL clinical samples, vortex and then centrifuge at a speed of 12 000 r/min and a centrifugation radius of 8.6 cm for 5 minutes. After centrifugation, discard the supernatant, add 20 μL of solution to resuspend the precipitate, and then add 10 μL of the resuspended precipitate into the PCR reaction solution(50 μL) for detection. The performance of the assay was evaluated from the aspects of the lower limit of detection, cross reactions, interfering substances and precision. Clinical samples (serum, urine and saliva samples) were collected from the Clinical Laboratory of Peking Union Medical College Hospital, and 1/5 of these samples were randomly selected to simulate clinical samples of ZIKV infection to further evaluate the performance of the method. Results The lower limit of detection(LOD) of the ZIKV assay for MR766 and ZKC2/2016 strains was 101 50% tissue culture infective dose(TCID50)/mL. A panel of 17 potential cross substances was tested and no cross-reactivity was detected. Potential interfering substances including bilirubin (15 mg/L), hemoglobin (100 mg/L), triglycerides (2000 mg/L), IgG (37.5 g/L) and EDTA-Na2(2.5 g/L) were evaluated and no obvious interference was observed. The intra-lots and inter-lots variations that were measured by coefficient of variation (CV), were lower than 10%. A total of 344 clinical samples from the Clinical Laboratory of Peking Union Medical College Hospital were collected; 73 simulated clinical samples of ZIKV infection were constructed. The detection results of 344 samples by this method were consistent with the expected results without false positives and false negatives. Conclusions The detection method for ZIKV has good specificity and repeatability. The detection results are not affected by interfering substances. The LOD meets clinical requirements, and the operation is simple. It can be used as a backup diagnostic tool for ZIKV and it needs to be further verified in real clinical samples.

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