Primers, probes, and amplicon sizes of the one-step real-time qRT-PCR assays.

<p>Primers, probes, and amplicon sizes of the one-step real-time qRT-PCR assays.</p

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

<div><p>Background</p><p>Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including <i>bunyaviruses</i>, <i>flaviviruses</i>, <i>filoviruses</i> and <i>arenaviruses</i>. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection.</p><p>Results</p><p>Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens.</p><p>Conclusions</p><p>Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs.</p></div

Amplification plots and standard curves of multiplex one-step real-time TaqMan RT-PCR assays.

<p>The multiplex one-step real-time TaqMan RT-PCR assays were tested using synthesized in vitro target viral RNA transcripts ranging from 10¹ to 10⁸ copies/µL. A PCR baseline subtractive curve fit view of the data is shown with relative fluorescence units (RFUs) plotted against cycle numbers. Standard curves generated from the Ct values obtained against known concentrations, the coefficient of determination (R²) and slope of the regression curve for each assay are indicated.</p

Detection limits of multiplex one-step real time qRT-PCR assays.

<p>Detection limits of multiplex one-step real time qRT-PCR assays.</p

Specificity analysis using in vitro transcribed viral RNAs.

<p>The number indicates Ct value determined from three replicates;</p><p>The minus represents a negative detection;</p

Evaluation of the multiplex real-time qRT-PCR assays using clinical specimens.

<p>*Patient types were confirmed by sera detection using the corresponding monoplex assays.</p

Specificity analysis using viral isolates and healthy human sera.

<p>The number indicates Ct value determined from three replicates;</p><p>The minus represents a negative detection;</p><p>*DENV1-4, 4 types of dengue virus, viral strains of Hawaii, New Guinea, H87 and H241 were used.</p

GLuc expression in replicon-defective transfected cells infected with varying PFUs of alphaviruses.

<p>BHK-21 cells were transfected with pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent, 6 h before mock-infection or infection with varying PFU levels of alphaviruses. GLuc activity was measured 44 h post-infection. Each data point represents the mean ±SEM of three independent experiments. RLU, relative light units.</p

Diagram of the generation of defective XJ-160 replicons.

<p>(A) The pVa-XJ replicon was constructed by inserting the XJ-160 virus genome into the eukaryotic expression vector pVAX1 and replacing the structural gene with the multiple cloning site (MCS) sequence. (B) The constructs containing the reporter gene (pVaXJ-EGFP or pVaXJ-GLuc) were generated by digesting the <i>enhanced green fluorescent protein</i> (<i>EGFP</i>) gene or <i>Gaussia luciferase</i> (<i>GLuc</i>) gene with the restriction enzymes <i>Fse</i>I and <i>Asc</i>I, and ligating them into the MCS of pVa-XJ. (C) The defective XJ-160 replicons (pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4) were produced by introducing an 1139-nt deletion mutation in the non-structural protein coding regions of pVaXJ-EGFP and pVaXJ-GLuc using <i>Acl</i>I digestion. The deleted regions of the XJ-160 genome in defective replicons are denoted by dotted lines and the non-structural protein gene deletion is designated by a “Δ”.</p

EGFP and GLuc expression in replicon-defective transfected cells infected with SINV (XJ-160).

<p>BHK-21 cells were transfected with pVaXJ-EGFPΔnsp4 or pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent, 6 h before mock-infection or infection with 1 MOI SINV (XJ-160). (A, B) EGFP expression was examined 44 h post-infection. Green color indicates EGFP, and blue color indicates nucleus. (C) GLuc activity was measured at different time points (from 6–68 h) post-infection. *<i>p</i><0.05, (pVaXJ-GLucΔnsp4+SINXJ160) vs. pVaXJ-GLucΔnsp4.</p