One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

Abstract

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8-100% sensitivity, 100% specificity, 86-89% efficiency and a limit of detection of 12-400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82-90% efficiency and limit of detection of 120-4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

Keywords: Gastroenteritis; Multiplex qRT-PCR; RotaTeq®; Rotarix®; Rotavirus genotyping.

Figure 1. Performance of 4 well multiplex qRT-PCR assay on lab cultured positive controls.

Well 1A (Xeno) - Amplification plots of Xeno-TR, RotaTeq®-HEX, Rotarix®-Cy5, and G12-FAM qRT-PCR on samples spiked with Xeno, RotaTeq® vaccine strain, Rotarix® vaccine strain and strain L26. Well 1B (MS2) - Amplification plots of MS2-TR, RotaTeq®-HEX, Rotarix®-Cy5, and G12-FAM assays on samples spiked with MS2, RotaTeq® vaccine strain, Rotarix® vaccine strain and strain L26. Well 2-Amplification plots of NSP3-TR, G9-HEX and G4-FAM on strains ST3 and 116E. Well 3- Amplification plots of G1-HEX, G3-Cy5 and P[4]-TR on strains Wa, P, and DS-1. Well 4- Amplification plots of P[6]-FAM, P[8]-Cy5 and G2-TR on strains ST3, Wa and DS-1. Amplification plots from FAM reporter dye are shown in green, amplification plots from HEX reporter dye are shown in orange, amplification plots from Texas Red reporter dye are shown in red and amplification plots from Cy5 reporter dye are shown in blue.

Figure 2. Performance of 4 well multiplex qRT-PCR assay on sequence confirmed clinical samples.

(A) Well 1- Amplification plots of Xeno-TR, RotaTeq®-HEX, Rotarix®-Cy5, and G12-FAM qRT-PCR on clinical samples spiked with Xeno. (B) Well 2- Amplification plots of NSP3-TR, G9-HEX and G4-FAM on clinical samples. (C) Well 3- Amplification plots of G1-HEX, P[4]-TR and G3-Cy5 assays on clinical samples. (D) Well 4- Amplification plots of G2-TR, P[8]-Cy5 and P[6]-FAM on clinical samples. Amplification plots from FAM reporter dye are shown in green, amplification plots from HEX reporter dye are shown in orange, amplification plots from Texas Red reporter dye are shown in red and amplification plots from Cy5 reporter dye are shown in blue.

Figure 3. Efficiency and limit of detection of Well 1 qRT-PCRs in multiplex reaction.

Amplification curves of (A) RotaTeq®-HEX, (C) Rotarix®-Cy5, (E) G12-FAM qRT-PCR using their respective 10-fold serial dilutions of dsRNA transcripts in multiplex reaction and the linear relationship between quantification cycle (Cq) and log transcript copy number per reaction (B) RotaTeq®-HEX, (D) Rotarix®-Cy5, (E) G12-FAM. Graphs showing the Cq value versus the log copy number were fitted with a regression line, and the slope for calculation of efficiency was obtained from the regression line.

Figure 4. Efficiency and limit of detection of Well 2 qRT-PCRs in multiplex reaction.

Amplification Curves of (A) G9-HEX, (C) NSP3-TR, (E) G4-FAM using their respective 10-fold serial dilutions of dsRNA transcripts in multiplex reaction and the linear relationship between quantification cycle (Cq) and log transcript copy number per reaction (B) G9-HEX, (D) NSP3-TR, (F) G4-FAM. Graphs showing the Cq value versus the log copy number were fitted with a regression line, and the slope for calculation of efficiency was obtained from the regression line.

Figure 5. Efficiency and limit of detection of Well 3 qRT-PCRs in multiplex reaction.

Amplification curves of (A) G1-HEX, (C) P[4]-TR, (E) G3-Cy5 using their respective 10-fold serial dilutions of dsRNA transcripts in multiplex reaction and the linear relationship between quantification cycle (Cq) and log transcript copy number per reaction (B) G1-HEX, (D) P[4]-TR, (F) G3-Cy5. Graphs showing the Cq value versus the log copy number were fitted with a regression line, and the slope for calculation of efficiency was obtained from the regression line.

Figure 6. Efficiency and limit of detection of Well 4 qRT-PCRs in multiplex reaction.

Amplification curves of (A) G2-TR, (C) P[8]-Cy5, (E) P[6]-FAM using their respective 10-fold serial dilutions of dsRNA transcripts in multiplex reaction and the linear relationship between quantification cycle (Cq) and log transcript copy number per reaction (B) G2-TR, (D) P[8]-Cy5, (F) P[6]-FAM. Graphs showing the Cq value versus the log copy number were fitted with a regression line, and the slope for calculation of efficiency was obtained from the regression line.