Improved detection of Lassa virus by reverse transcription-PCR targeting the 5' region of S RNA

Abstract

The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1994), which prompted us to revise the protocol. The design of the new assay, the GPC RT-PCR/2007 assay, took into account 62 S RNA sequences from all countries where Lassa fever is endemic, including 40 sequences generated from the strains in our collection. The analytical sensitivity of the new assay was determined with 11 strains from Sierra Leone, Liberia, Ivory Coast, and Nigeria by probit analysis; the viral loads detectable with a probability of 95% ranged from 342 to 2,560 S RNA copies/ml serum, which corresponds to 4 to 30 S RNA copies/assay. The GPC RT-PCR/2007 assay was validated with 77 serum samples and 1 cerebrospinal fluid sample from patients with laboratory-confirmed Lassa fever. The samples mainly originated from Liberia and Nigeria and included strains difficult to detect in the assay of 1994. The GPC RT-PCR/2007 assay detected virus in all clinical specimens (100% sensitivity). In conclusion, a new RT-PCR assay, based in part on the protocol developed by Demby et al. in 1994, for the detection of Lassa virus is described. Compared to the assay developed in 1994, the GPC RT-PCR/2007 assay offers improved sensitivity for the detection of Liberian and Nigerian Lassa virus strains.

FIG. 1.

Binding site of primer LVS-339-rev in different Lassa virus strains. The primer sequence is indicated at the top of the alignment in the 5′ to 3′ orientation. The positions are numbered according to the S RNA of Lassa virus strain Josiah. The primer contains two wobble bases (underlined), and dots at these positions in the alignment represent identities to either of the two possible bases (M = A or C, K = G or T). Experimental evidence for the ability of the GPC RT-PCR/2007 assay to detect one or several of the strains is summarized on the right: probit (P), the in vitro transcript of S RNA was used to determine the 95% detection limit (Table 2); virus culture (VC), the isolate was detected in the supernatant of the virus culture (Table 1); serum (S) and CSF, virus was detected in serum or CSF from infected humans or animals. The strain Nig09-OSPMH sequences were kindly provided by S. Omilabu (unpublished data).

FIG. 2.

Analytical sensitivity of the GPC RT-PCR/2007 assay determined by probit analysis. The analysis was performed with pooled data, shown in Table 2, for all 11 virus strains. Filled squares, experimentally determined fractions of positive results (y axis) at a given RNA concentration (x axis); solid line, regression curve for the probability of obtaining a positive RT-PCR result at a given concentration of Lassa virus RNA in plasma; right and left dotted lines of the regression curve, the 95% confidence interval; cop, number of copies.

FIG. 3.

Detection of Lassa virus in Liberian serum samples by using the GPC RT-PCR/2007 assay. RNA was extracted from serum and tested undiluted (ud) and at a 1:10 dilution. The results obtained by the GPC RT-PCR/1994 assay according to the protocol published in 2002 (4) for routine diagnosis are shown below the agarose gel. Samples Lib05-1580/121 and Lib05-2406/129 were positive by virus culture. NTC, nontemplate control; Pos. Control, positive control. The numbers on the right are in base pairs.