Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection

Abstract

The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log copies/μL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.

Yellow Fever Virus: Diagnostics for a Persistent Arboviral Threat

“…A number of molecular tests for YFV have been described in the literature, including conventional RT-PCR (41,(60)(61)(62), real-time RT-PCR (rRT-PCR) with hydrolysis probes or SYBR green (39,41,42,(63)(64)(65)(66)(67), multiplex rRT-PCR (68)(69)(70), and isothermal methods (64,(71)(72)(73). Representative real-time and isothermal assays are shown in Table 1.…”

“…YFV detection may be improved in areas of endemicity if testing is performed in multiplex with related pathogens that cause an acute febrile illness. However, few multiplex rRT-PCRs that specifically detect YFV have been developed (68)(69)(70). Of those, only the assay by Rojas et al detects another related human pathogen (DENV) and has been developed for and evaluated with clinical samples (70).…”

“…However, few multiplex rRT-PCRs that specifically detect YFV have been developed (68)(69)(70). Of those, only the assay by Rojas et al detects another related human pathogen (DENV) and has been developed for and evaluated with clinical samples (70). Different isothermal methods for YFV detection have been developed and evaluated (Table 1) (64,(71)(72)(73).…”

What Does the Future Hold for Yellow Fever Virus? (II)

“…Until the beginning of the 21st century, molecular YFV diagnostics was achieved partly by conventional reverse transcription PCR (RT-PCR) [ 203 , 204 , 205 ]. Subsequently, YFV-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) [ 206 , 207 ], real-time recombinase polymerase amplification (RT-RPA) [ 208 ] and real-time RT-PCR assays using either intercalant dyes such as SYBR Green I [ 209 , 210 ] or TaqMan (hydrolysis) probes [ 211 , 212 , 213 , 214 , 215 , 216 , 217 , 218 , 219 , 220 ] were developed. Very recently, a Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) was developed for DENV and Zika virus (ZIKV) detection by using the RNA-targeting CRISPR-associated enzyme Cas13 [ 221 , 222 ].…”

A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

“…For rapid and accurate diagnosis of dengue virus in early phases of acute infections, detection of DENV RNA is recommended as the most sensitive and specific method to diagnose dengue in the acute phase of the illness by WHO [9]. After the first developed nucleic acid detection test for dengue diagnosis over 20 years ago [15], there have been a number of RT-PCR assays for DENV detection and serotype identification with high sensitivity and specificity [16][17][18][19][20]. Combined with automated RNA extraction protocols, RT-PCR can potentially facilitate fast and accurate diagnosis of dengue suspected cases with a single sample obtained during the patient's first visit to the clinician.…”