Optimization of qRT-PCR assay for zika virus detection in human serum and urine

Abstract

Zika Virus (ZIKV) is a mosquito-borne flavivirus that the World Health Organization (WHO) declared a global concern due to the severity of infection. This study focuses on determining the level of detection of ZIKV RNA in human serum and urine. Known amounts of Zika virus were added to uninfected human serum and urine samples. Different reverse transcriptases were compared to select the optimal enzyme for this application. Zika RNA in these samples was then quantified with qRT-PCR to determine the lower limit of detection in these fluids and to construct a standard curve. Student's t-test of paired samples was used in order to identify statistical differences. The SuperScript III enzyme was able to produce more ZIKV cDNA when compared to PrimeScript. Zika virus RNA was found to be detectable at lower levels (2.5 PFU/mL) in urine than in serum (250 PFU/mL) when using SuperScript III. This study demonstrates how the selection of both the human clinical specimen, and the reverse transcriptase enzyme involved in the molecular detection of ZIKV by quantitative real-time polymerase chain reaction (qRT-PCR), play an important role in enabling improved detection of the virus.

Keywords: Clinical samples; Limit of detection (LOD); Reverse transcriptase; Zika Virus (ZIKV); qRT-PCR.

Figure 1.. ZIKV detection by qRT-PCR at different concentrations of the virus in human serum and filtered (“F”) urine.

qRT-PCR was carried out using two reverse- transcriptase enzymes: SuperScript III (SSIII) represented in blue for filtered urine (F-Urine) and in orange for serum samples, and PrimeScript (PS) represented in grey and yellow for F-Urine and serum respectively. The experiment was performed in triplicate with the corresponding standard deviation measurement, and the addition of a negative control (NTC).

Figure 2:. Reverse transcriptase efficiency for ZIKV detection.

Side-by-side comparison of the effect that reverse-transcriptase SSIII (orange and yellow) or PS (blue and grey) had on ZIKV detection in serum (A) and filtered urine (B) by qRT-PCR. A two-tailed Student’s t-test of the paired means was used to calculate the p- values when comparing the two enzymes in the same matrix.

Figure 3:. ZIKV detection in ZIKV-spiked clinical sample types.

(A) Ct values corresponding to ZIKV detection by qRT-PCR in filtered urine (blue and grey bars) and serum (orange and yellow bars) using either the SSIII (upper panel) or the PS (lower panel) enzyme. A two-tailed Student’s t-test of the paired means was used to calculate the p-values when comparing the each enzyme in both matrices. (B) Effect of filtration (“F”) and spinning (“Sp”) before RNA isolation for ZIKV identification.

Figure 4:. Sensitivity of the qRT-PCR assay.

(A) Quantification and standard curve of number of molecules of ZIKV RNA/μL. Ct values were generated by qRT-PCR of extracted 10-fold serial diluted RNA from ZIKV stock. The standard curve was generated with 10-fold serial dilutions of ZIKV RNA. Ct values obtained are represented against the log of the number of molecules of ZIKV RNA. (B) Quantification and standard curve of ZIKV infectious particles. Ct values were generated by qRT-PCR of extracted 10-fold serial diluted RNA from ZIKV stock. The standard curve was generated with 10-fold serial dilutions of ZIKV RNA. Ct values obtained are represented against the log of the quantity of infectious viral particles (PFU/mL).

Figure 5:. ZIKV detection in ZIKV-infected individual clinical samples.

Number of viral molecules/μL were calculated through qRT-PCR after viral RNA isolation and cDNA synthesis with two different RTases (PS, SSIII) in serum and urine samples. ZIKV was detected in one out of three urine samples tested, with sera from all three patients being under the limit of detection for the assay.