Preparation of MS2-based nanoparticles as control and standard materials for the molecular detection of dengue virus serotypes

Highlights

• We Prepare DENV-1 to 4 nanoparticles as control and standard for the first time for detection of dengue virus.

• Performance for detection of dengue virus is acceptable in China.

• The application of the nanoparticles for quantitative detection of DENV is reliable and promising.

Abstract

To quantify dengue virus (DENV) and evaluate the performance of clinical laboratories using quantitative real-time polymerase chain reaction (qRT-PCR) assays, we constructed high-efficiency expression systems to produce DENV-1 to 4 nanoparticles and assessed their suitability as standard and control materials in 20 laboratories across China. Targeted gene sequences of DENV-1 to 4 were synthesized and inserted into pACYC-Duet 1-MS2 recombinant plasmids to generate corresponding nanoparticle expression systems. After collection, verification, and quantification by digital PCR (dPCR), DENV-1 to 4 nanoparticles were prepared as control and standard materials. Five positive and three negative samples of each DENV serotype in every panel were used for assessing the performance of the participating laboratories across China, as well as standard materials for the quantitative detection of DENV using qRT-PCR assays. The accuracy, sensitivity, and specificity of qRT-PCR used by the 20 evaluated laboratories were 89.6 (569/635), 85.1 (336/395), and 97.1% (233/240), respectively. Overall, sixteen (80.0%) laboratories were qualified in detecting DENV, among which five (25.0%) were designated as “competent”, eleven (55.0%) were defined as “acceptable”, and four (20%) were considered to be “improvable”. The results generated from the DENV standard samples were significantly positively correlated with those generated by dPCR (r² = 0.8698, P < 0.001). In summary, DENV nanoparticles could potentially be used as controls for improving the performance of laboratories and as standards for the quantitative detection of DENV.

Introduction

Dengue is the most rapidly spreading mosquito-borne virus in the world. The latest world-wide survey indicated 390 million cases of dengue infections per year (Bhatt et al., 2013). Apart from indigenous cases in tropical and sub-tropical regions, recently imported cases from epidemic areas could also potentially cause a dengue pandemic. In 2014, part of the dengue epidemic of Guangdong in China was caused by cases imported from Malaysia and Singapore, that then spread to 20 other cities (Huang et al., 2016a), even as far as Japan (Quam et al., 2016). Imported cases were also reported in America (Moncayo et al., 2015), Italy (Quam et al., 2015), and Pakistan (Wesolowski et al., 2015). In consideration of the threat to human health and the substantial economic cost caused by dengue virus (DENV) (Guzman et al., 2010, Martina et al., 2009, Shepard et al., 2013), effective measures should be taken to control its transmission. These measures should include epidemiological surveillance by governments (De Simone et al., 2004, Guo et al., 2016, WHO, 2009), and efficient and accurate detection of DENV in laboratories (Guo et al., 2016, WHO, 2009).

Various DENV serotypes are transmitted to humans through the bites of infected Aedes mosquitoes, principally Ae. Aegypti (WHO, 2009). Their genomes code for a core protein, precursor membrane protein, envelope protein, and nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) (Guzman et al., 2010). Various real-time polymerase chain reaction (RT-PCR) assays have been developed for DENV detection and serotype identification (Chien et al., 2006, De Simone et al., 2004, Hue et al., 2011, Johnson et al., 2005, Muñoz-Jordán et al., 2009). However, when detecting the DENV, several factors, such as nucleic acid extraction techniques, detection methods, instruments, commercial assays used, and disparate individual operators can cause variations in a laboratory’s performance. This makes results obtained from different laboratories non-comparable. Furthermore, many RT-PCR assays have not been validated for accuracy and quality; approximately 10% of published methods show optimal performance, while more than 80% lack sensitivity and/or specificity information (Domingo et al., 2010). Notably, the quantitative (q)RT-PCR assay is highly sensitive to amplicon contamination, and false-positive results may occur. Meanwhile, RNA degradation in the detection process of DENV can result in false-negative results and affect the laboratory performance.

Although there are various problems associated with detecting DENV, no effective measures have been taken to monitor the performance of laboratories and detection assays for this virus in China because of the difficulty in obtaining sufficient clinical samples to prepare control materials. Although quantification of the virus in clinical samples is helpful for improving the ability of laboratories to detect the virus, there are few studies on quantification of the DENV in the world. Considering the above conditions, in this study we constructed DENV-1 to 4 nanoparticle expression systems to produce corresponding MS2-based virus-like particles. We then assessed their suitability as standards for the quantitative detection of DENV-1 to 4, as well as to serve as control materials for assessing the performance of laboratories using qRT-PCR assays across China.