Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis

Highlights

• An RT-PCR combined high resolution melting analysis was established.

• The assay was probe-free and sensitive.

• It was applied for the detection of four diarrhea-causing viruses.

Abstract

Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10⁰, 10², 10⁰ and 10³ copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens.

Introduction

Diarrhea is one of the most common public health issues worldwide. Though spontaneous cure occurs in approximately four days, a few patients show various symptoms for weeks. More seriously, some patients may even develop irritable bowel syndrome if left untreated [1]. Therefore, rapid and sensitive diagnostic methods are urgently needed to help physicians make faster and better treatment decisions for those patients who suffered from diarrhea.

The causative agents responsible for acute diarrhea are bacteria, parasites or viruses, among which rotaviruses, astroviruses, noroviruses and sapoviruses are the most important pathogens and accounts for majority of the viral related diarrhea cases [2], [3], [4], [5]. It was reported that these four viruses caused up to 39%, 26%, 26% and 9% of diarrhea cases, respectively [6], [7], [8]. Moreover, more and more co-infections were detected [9], [10]. Traditional detection methods for these viruses are electron microscopy, northern blot, serological tests or antigen-detection assay, all of which are either low-sensitive, labor-intensive or time-consuming [11]. In contrast, molecular diagnostic methods could provide specific and sensitive detection for these pathogens. In previous studies, multiplex RT-PCR assays were used for the detection of rotaviruses, astroviruses, noroviruses, sapoviruses and other related viruses [12], [13], [14], [15]. However, expensive molecular probes or contamination-prone gel electrophoreses were needed during or after amplification in these methods. Recently, probe-free high resolution melting analysis (HRMA) which uses novel saturation fluorescent dyes (LCGreen I or EvaGreen) for the direct characterization of PCR amplicons in a closed system has been developed for rapid and high-throughput mutation analysis [16], [17], genotyping [18], [19], [20], and pathogen detection [21], [22], [23], [24], [25]. For diarrhea-causing viruses detection, Etsuko Tajiri-Utagawa et al. developed a HRMA assay for the routine detection and genotyping of noroviruses [26], while Akihiko Hata et al. applied HRMA for the detection and differentiation of human astroviruses [27]. However, to date, there were no reports on the detection of rotaviruses or sapoviruses by HRMA based assay.

In the current study, a probe-free RT-PCR combined HRMA assay was established for the sensitive detection of these four major diarrhea-causing pathogens. Specificity and sensitivity of the assay were determined and compared with both conventional RT-PCR and commercial real-time RT-PCR. Real stool samples were used to evaluate the efficacy of the assay for its potential clinical application.