A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test

Abstract

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

Figure 1

Sequence alignment of the degenerate primers with the targeted region of the nucleoprotein gene of 24 isolates of 14 different lyssavirus species.

Figure 2

Kinetic profile of RABV infection in mice determined by generic lyssavirus qRT-PCR, FAT and virus titration. Mice were infected with 3 × 10² TCID₅₀ of RABV (CVS-11) by intranasal inoculation and sacrificed 0 to 10 days later. The brain was collected for analysis. The course of (a) viral RNA by generic lyssavirus qRT-PCR, (b) load of viral antigen (FAT), (c) infectious virus (RTCIT), and (d) correlation of Cq values between the generic lyssavirus and RABV-specific qRT-PCR are presented. The correlation between the Cq values obtained by both qRT-PCR methods was excellent (Pearson's correlation coefficient r = 0.9773, P < 0.0001).

Figure 3

Melting peaks obtained for 7 lyssavirus species. The melting temperatures for RABV, MOKV, LBV, DUVV, EBLV-1, -2, and ABLV were, respectively, 77°C, 77.5°C, 76°C, 76.5°C, 78.5°C, 78°C, and 76.5°C. No primer dimers were observed.

Figure 4

Analytical sensitivity of the monospecific RABV and the generic lyssavirus qRT-PCR for RABV (CVS-11) (a) and EBLV-1 (b). Six independent runs with each time two repeats were performed per virus dilution (10-fold serial dilution). There was an excellent linear regression between the load of infectious virus, determined by virus titration, and the Cq value for RABV and EBLV-1 (regression coefficient of 0.965 and 0.989, resp.). For both qRT-PCR methods, the limit of detection of RABV and EBLV-1 was ≤ 100 TCID₅₀. The Cq remained undetectable in the negative control samples.*Mean and standard deviation are calculated based on the runs/repeats with a positive signal (Cq ≤ 40). ND = not determined.