Sequence optimized diagnostic assay for Ebola virus detection

Abstract

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013-2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Figure 1

Sequence mismatches identified among EBOV variants. (A) Multiple EBOV variants including Mayinga (GenBank #NC_002549), Kikwit (#KR867676), Makona Sierra Leone (#KM233075), Makona Mali (#KP260799), Gabon (#KC242792), and Luebo (#KC242785) were aligned. Shown is the assay target region with the forward and reverse primers indicated by the outer primers and the probe by the inner arrows. (B) Primer and probe sequences of primers used throughout the manuscript. Note the color schemes for the primers and probe in (A), (B) correspond to primer matches with the respective EBOV variant. For example, the green R2079 (G14A, G18A) matches with EBOV Luebo and is shown in (A) as the green reverse primer. The R2079 (G18A) primer contains a single, isolate variant indicated by a * in the reverse primer of EBOV Luebo in (A).

Figure 2

Primer and probe mismatch analysis identified impact of sequence mismatches on target detection. (A) EBOV variants Kikwit, Mayinga, Makona, and Luebo were tested by RT-qPCR using different primer and probe combinations identified in Fig. 1. Reported are the Cq values for each assay combination tested with nucleic acid from each EBOV variant. Samples were tested in triplicate and error bars indicate standard deviation. The bar colors correspond to the primers and probe shown in Fig. 1A. (B) Changes in real-time PCR signal (ΔCq) was calculated with regard to the Ebo-TM assay. Differences in the primer and probe with regard to the template are shown as a match, a single variant, or a double variant. Significance was determined using a t-test (Holm-Sidak method), and a comparison was considered significant if the difference was less than 0.05 (*p < 0.05, **p < 0.01).

Figure 3

Preliminary LOD identify improved sensitivity for the sequence optimized EBOV assay. Preliminary LODs were determined using serial dilutions of EBOV variants Kikwit (A), Mayinga (B), Makona (C), and Luebo (D). Extracted nucleic acid was serially diluted 1:10 in nuclease-free water and assayed by the Ebo-TM assay and the sequence optimized EBO-TM2 assay. Samples were run in triplicate, and the preliminary LOD was lowest concentration where all three replicates were positive. Error bars indicate the standard deviation.