Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR

Abstract

Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5'-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5'-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5'-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The >or=95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.

FIG.1.

Target regions of newly designed primers and 5′-nuclease probes. All sequences of the target regions available at GenBank were aligned. Each type of sequence variation within this region is shown once, with its frequency on the right. GenBank accession numbers of representative sequences for each type of variation are indicated on the left. Primer and probe sequences as well as their designations are given above the alignment. The antisense primer sequences are reverse complement (rc). If two primers were chosen for one binding site, both primer variants are shown, with variable positions underlined. Boldface nucleotides in the DENV alignment indicate positions which are shared by the binding site of the sense strand probe and the antisense primer. An asterisk indicates the end of a GenBank entry, and a slash indicates a gap in the alignment.

FIG. 2.

Sensitivity of one-step RT-PCR in the absence (−) and presence (+) of SybrGreen. The dye was lyophilized at the bottom of the capillary. In vitro-transcribed LASV RNA was amplified in a LightCycler as described in Materials and Methods. The PCR products were visualized in ethidium bromide-stained agarose gel. Input RNA copy numbers are indicated above the lanes. The negative control (neg) contained RNA extracted from negative plasma.

FIG. 3.

Determination of RT-PCR detection limits by probit regression analysis. Negative human plasma was spiked with defined amounts of in vitro-transcribed RNA and prepared three times in parallel. Each RNA preparation was amplified two times in parallel, resulting in six replicate RT-PCRs per RNA test concentration. The experimentally determined fraction of positive reactions (n_positive/n_tested) (y axis) at the corresponding RNA test concentration (copies per milliliter of plasma) (x axis) is shown by squares. The calculated regression curves (middle curve) indicate the probability (y axis) of obtaining a positive result at any RNA concentration. The 95% confidence intervals for this probability are shown by curves at the left and right of the middle curve. The RNA concentration at which a positive result is achieved with a probability of 95% is given in the text.

FIG. 4.

Detection and quantification of VHF pathogens in clinical specimens. RNA was prepared from serum samples of VHF patients (see Materials and Methods for details on the patients). Sera from patients with Ebola fever were diluted 1:1,000 to overcome inhibition, and those from Lassa fever patients were diluted 1:100 to save serum. A log₁₀ dilution series of in vitro-transcribed RNA was amplified as a standard; the copy number per milliliter of plasma is shown at the curves. The graphs show the real-time detection of the specific PCR products by fluorescence (y axis) (F1 detection wavelength or F1/F2 ratio) dependent on the PCR cycle number (x axis). Despite the low plateau level in some reactions with low template copy numbers, the signals are clearly positive in both melting curve analysis and agarose gel analysis. The insert in the upper left corner of each graph depicts the standard curve (x axis, RNA concentration; y axis, cycle number at first detection of PCR product). For RVFV, no clinical sample was available (the graph is shown to illustrate the technical performance of the PCR). The specimens from the patients with Ebola hemorrhagic fever were diluted 1:1,000 to reverse RT-PCR inhibition, which probably resulted from extensive hemolysis.