Synthesis of the HTNV cRNA

Viral RNA was extracted from a HTNV virus stock, and cDNA was synthesized using viral RNA as the template and a combined, reverse transcription-polymerase chain reaction (RT-PCR). The length of the amplified cDNA products was 516 bp, which was the expected size (Figure 1). HTNV cRNA was synthesized in vitro using T7 RNA polymerase and the cDNA as templates. The size of the HTNV cRNA was 499 bp. The absorbance of HTNV cRNA was measured using a spectrophotometer, and the concentration was found to be 150.84 ng/μl. The copy number of the HTNV cRNA was thus calculated as 5.27×10¹¹ copies/µl.

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Figure 1. Agarose gel electrophoresis of HTNV cDNA and cRNA.

(A) cDNA with a length of 516 bp was synthesized by reverse-transcription and subsequently amplified by PCR. Marker: 50 bp DNA ladder. (B) HTNV cRNA with a length of 499 bp was synthesized using the cDNA as template by in vitro transcription. Marker: DNA ladder DL2000.

https://doi.org/10.1371/journal.pone.0081525.g001

Standardization of the SYBR Green Ⅰ-based qRT-PCR assay

Using 10-fold serial dilutions of the synthesized HTNV cRNA templates, SYBR Green Ⅰ-based one-step qRT-PCR was performed to establish a standard curve. The external standard curve was generated by plotting the Ct values versus serial dilutions of the HTNV cRNA templates ranging from 1×10⁸ to 1×10³ copies/µl. The observed linearity was good for the standard curve over a wide range of HTNV cRNA dilutions in triplicate tests, and the correlation coefficient (R²) was 0.998. The slope of the curve was -3.273, and the amplification efficiency of the assay was 102.1% based on the slope of the exponential phase in the amplification chart. The cycle intervals of qRT-PCR between each dilution ranged from 2.9 to 3.8 cycles (Figure 2).

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Figure 2. The HTNV cRNA amplification chart and standard curve.

(A) 10-fold serial dilutions of the HTNV cRNA standards ranging from 1×10⁸ to 1×10³ copies/µl were amplified by qRT-PCR assay. (B) The HTNV cRNA standard curve was plotted by Ct values versus the HTNV cRNA log copy numbers in triplicate tests.

https://doi.org/10.1371/journal.pone.0081525.g002

Optimization of the SYBR Green Ⅰ-based qRT-PCR assay

The annealing temperature of the assay was optimized at 57°C using a gradient of annealing temperatures from 51°C to 61°C. The optimal amount of input cRNA templates was 0.2 μg per reaction, and each primer in the reaction tube was used at a final concentration of 0.4 µM.

Specificity of the SYBR Green Ⅰ-based one-step qRT-PCR

This one-step qRT-PCR assay was highly specific for detecting HTNV because RNA samples from SEOV (also in the genus Hantavirus) and other viruses (HBV, HCV, and HIV) were not amplified during the assay. Melting curve analysis on the qRT-PCR products showed that no primer-dimers or non-specific products were formed, and only a single, sharp peak at 84°C was visible in the melt peak chart for HTNV cRNA and serum samples from HFRS patients (Figure 3). Only HTNV was amplified when SEOV and HTNV were tested simultaneously using the assay, and no sharp peak was visible for the SEOV qRT-PCR products in the melting curve chart (Figure 4).

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Figure 3. Melting peaks of the SYBR Green Ⅰ-based one-step qRT-PCR products.

(A) Melting peaks of the qRT-PCR products obtained from in vitro transcribed HTNV cRNA ranging from 1×10⁸ to 1×10³ copies/µl. Only a single sharp peak at 84°C was visible in the melt peak chart. (B) Melting peaks obtained from serum samples of HFRS patients. All melting curves in A and B have similar shapes.

https://doi.org/10.1371/journal.pone.0081525.g003

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Figure 4. Amplification chart and melt peak chart of HTNV and SEOV.

(A) Different dilutions of HTNV and SEOV RNA were tested using the SYBR Green Ⅰ-based one-step qRT-PCR assay, and only HTNV RNA was amplified. (B) A single sharp peak at 84°C in the melt peak chart indicated that only specific products of HTNV was obtained.

https://doi.org/10.1371/journal.pone.0081525.g004

Sensitivity of the SYBR Green Ⅰ-based one-step qRT-PCR

The sensitivity of the qRT-PCR assay was evaluated by measuring 10-fold serial dilutions of the HTNV cRNA standards ranging from 1×10⁶ to 1×10^-1 copies/µl in duplicate. The results indicated that the qRT-PCR assay could detect the HTNV RNA at levels as low as 10 copies/µl. The 237-bp amplicons of the qRT-PCR products were observed by electrophoresis on a 2% agarose gel (Figure 5).

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Figure 5. Sensitivity of the SYBR Green Ⅰ-based one-step qRT-PCR assay.

(A) Serial dilutions of the HTNV cRNA standards ranging from 1×10⁶ to 1×10^-1 copies/µl were amplified using the SYBR Green Ⅰ-based one-step qRT-PCR assay in duplicate, and the minimum detection limit of the qRT-PCR assay was 10 copies/µl. (B) The qRT-PCR products were electrophoresed on a 2% agarose gel. Marker: DNA ladder of DL2000.

https://doi.org/10.1371/journal.pone.0081525.g005

Intra- and inter-assay variations

The reproducibility of the qRT-PCR assay was established using the Ct values that were obtained from testing the HTNV cRNA standards in triplicate within each run (intra-assay) and in three different runs (inter-assay). The standard deviation (SD) and coefficient of variation (CV) values for the intra-assay ranged from 0.177 to 0.770 and from 0.68% to 3.00%, respectively. The SD and CV of the inter-assay were slightly higher than the intra-assay and ranged from 0.138 to 1.126 and from 0.86% to 3.21%, respectively, thus demonstrating good reproducibility (Table 1).

Assay performance on clinical samples

To evaluate the usefulness of the assay for clinically detecting HTNV RNA, the protocol was applied to serum samples of 27 HFRS patients who had been diagnosed by clinical manifestations and laboratory examination. All 27 HFRS patients showed positive serum IgM and/or IgG test results for hantavirus antibodies. The peripheral blood were collected during the febrile phase of the illness (3-7 days after onset of fever), and the copy numbers of the HTNV RNA in the serum samples ranged from 2.06×10¹ to 1.95×10⁵ copies/µl (Table 2). The copies of the HTNV RNA in the serum samples from HIV, HBV and HCV patients and normal controls were all below the level of detection.

Cells and viruses

The HTNV strain 76-118 and SEOV strain R22 were used in this study. HTNV was passaged in Vero-E6 cells (American Type Culture Collection, CRL 1586) that were grown in MEM/EBSS medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Rockville, MD, USA). HTNV infection was monitored by indirect immunofluorescence assay (IFA) using mouse anti-NP mono-antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (SouthernBiotech, Birmingham, AL, USA). After 10 days of incubation, the cell monolayer was lysed by three freeze-thaw cycles and centrifuged at 4,000 g for 10 min. The supernatant was aliquoted and stored at -80 °C.

Viral RNA extraction

The RNA of hantaviruses (HTNV and SEOV) was extracted from collected virus stocks according to the instructions in the QIAamp Viral RNA Mini Handbook (Qiagen, Hilden, Germany). The viral RNA (60 µl) was eluted and stored at -80°C until use, and the concentration and quality of the eluted RNA were measured by UV absorbance at 260 nm and 280 nm using a Thermo Scientific NanoDrop^TM Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Clinical specimens and Ethics Statement

27 HFRS patients, 3 HBV patients, 3 HCV patients, 3 HIV patients and 4 normal controls were recruited from the Center of infectious diseases in the Tangdu Hospital from November, 2012 to January, 2013, and enrolled in the study. The diagnosis of HFRS was based on clinical manifestations and confirmed by the detection of hantavirus IgM/IgG antibodies using Hantavirus IgG/IgM Combo Test Card (Boson Biotech, Xiamen, China). Samples of peripheral blood from 27 HFRS patients were collected during the febrile phase of the illness (3-7 days after onset of fever) and stored at -80°C until use. HFRS disease severity was classified on the basis of clinical manifestations and laboratory parameters used in the diagnostic criteria for HFRS in China as mild, moderate, severe, and critical. The viral RNA extraction procedure was same as those described above. The clinical information about the HFRS patients, such as the days after onset of fever, the severity of the disease, was also collected and documented. The clinical study protocol was approved by the Ethics Committee of Fourth Military Medical University, and written informed consent was obtained from all enrolled subjects.

Primer design and modification for the synthesis of HTNV cRNA

Alignments of HTNV S segment sequences encoding the NP of strain 76-118 (M14626.1) and other strains (A16 (AF288646.1), A9 (AF329390.1), and 84FLi (AY017064.1)) were performed based on the epidemiological data on HTNV epidemic strains in the Xi’an district, Shaanxi Province [4]. The primers 5’-ATCCTTTGTCGTCCCGATACTT-3’ (position of 417 to 438) and 5’-ATAGCCTTTGACTCCTTTGTCTCC-3’ (position of 864 to 887) were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The forward primer was modified by incorporating a T7-promoter sequence (5'-TAATACGACTCACTATAGGG-3') on its 5'-end of the primer. The reverse primer was modified by adding an oligo-(dT) to its 3'-end (File S1). The length of the constructed HTNV cRNA standards was 499 bp.

Amplification of cDNA template by conventional RT-PCR

The eluted HTNV RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The first-strand cDNA master mix (20 µl) was prepared as follows: 2 µl of template RNA was mixed with 1 µl of random primer (0.2 μg/μl), 4 µl of 5 × Reaction Buffer, 1 µl of RiboLock RNase Inhibitor (20 u/µl), 2 µl of 10 mM dNTP Mix, 1 µl of RevertAid M-MuLV Reverse Transcriptase (200 u/µl) and 9 µl of nuclease-free water. The reverse transcription reaction mixture was incubated for 5 min at 25°C followed by 60 min at 42°C.

The following PCR amplification was performed in 20 µl of mixture composed of 10 μl of DreamTaq^TM PCR Master Mix (2 ×) (Thermo Fisher Scientific, Waltham, MA, USA), 1 μl of each modified primer (10 μM), 2 μl of RT products and 6 μl of nuclease-free water. The PCR reaction was performed using a Bio-Rad MyCycler thermal cycler (Bio-Rad, Hercules, CA, USA) with the following parameters: 95°C at 2 min followed by 35 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C and a final extension at 72°C for 7 min. After amplification, the PCR products were electrophoresed in a 2% agarose gel (Biowest, Madrid, Spain) and visualized using a Gel Doc System (Bio-Rad, Hercules, CA, USA).

Precipitation of PCR products and in vitro transcription

The PCR products were precipitated with 3 M NaOAc and 100% ethanol and then washed once with 70% ethanol. Subsequently, the products were submitted to in vitro transcription using T7 RNA polymerase (TaKaRa Biotechnology, Dalian, China) to synthesize cRNA in accordance with the manufacturer’s instruction. HTNV cRNA was processed twice with DNase I (TaKaRa Biotechnology, Dalian, China) to remove residual DNA, and this step was followed by chloroform/isoamyl alcohol/ethanol precipitation. The HTNV cRNA transcripts were finally dissolved in 40 µl of RNase-free water, electrophoresed in a 2% agarose gel (Biowest, Madrid, Spain) and visualized by ethidium bromide. The HTNV cRNA stock solution was serially diluted and stored at -80°C until use.

Quantification of the HTNV cRNA

The concentration and quality of the HTNV cRNA was measured by UV absorbance at 260 nm and 280 nm in duplicate using a Thermo Scientific NanoDrop^TM Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The copy number of the HTNV cRNA standards was calculated using the following formula [22]: RNA copy number (copies/μl) = RNA concentration (g/μl) × 6.02 × 10²³ (copies/mol)/345 × RNA length (b).

SYBR Green Ⅰ-based one-step qRT-PCR assay

qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China). Briefly, 2 µl of each HTNV cRNA standard was mixed with 12.5 µl of 2 × One-Step SYBR RT-PCR Buffer, 1 µl of PrimeScript One-Step Enzyme Mix 2, 1 µl of each primer (0.4 µM), and 7.5 µl of RNase-free water in 8-strip tubes (BIOplastics, Landgraaf, Netherlands). The forward primer, 5’-AAGCATGAAGGCAGAAGAGAT-3’ (position of 594 to 614), and the reverse primer, 5’-TAGTCCCTGTTTGTTGCAGG-3’ (position of 811 to 830), which amplified a 237 bp fragment, were designed using the Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The sequences of the primers were compared to sequence databases using the program BLAST on the NCBI website, and the primer pairs were synthesized and purified by Sangon (Sangon Biotech, Shanghai, China). The thermal cycling conditions comprised reverse transcription at 42°C for 5 min and 95°C for 10 s, followed by 40 cycles of PCR amplification at 95°C for 5 s and 57°C for 30 s. After amplification, a melting curve analysis program was used to verify the authenticity of the PCR products by their specific melting temperatures (Tm) in accordance with the manufacturer’s instructions.

Generation of a standard curve using the SYBR Green Ⅰ-based one-step qRT-PCR assay

To construct a standard curve, 10-fold serial dilutions of the HTNV cRNA standards spanning 1×10⁸ to 1×10³ copies/µl in Easy Dilution (TaKaRa Biotechnology, Dalian, China) were used and tested in triplicate. Standard curve was plotted as the mean Ct values versus the log HTNV cRNA copy numbers. Regression analysis, standard curve slopes and amplification efficiencies were calculated using automated software (Bio-Rad iQ5 2.0 Standard Edition Optical System Software).

Optimization of the SYBR Green Ⅰ-based one-step qRT-PCR assay

Different parameters, such as the gradient of annealing temperature (ranging from 51 to 61°C), the gradient of primer concentrations and amounts of cRNA templates in 25 µl of reaction volume, were tested for the optimization of the amplification of specific HTNV products.

Assay specificity

To investigate the specificity of the SYBR Green Ⅰ-based one-step qRT-PCR assay, SEOV and other viruses (HBV, HCV, and HIV) were subjected to qRT-PCR with the same amplifying primers and reaction conditions.

Assay sensitivity

The detection limit of the qRT-PCR was determined using 10-fold serial dilutions of the HTNV cRNA ranging from 1×10⁶ to 1×10^-1 copies/µl in duplicate.

Assay reproducibility

Serial dilutions of the HTNV cRNA standards ranging from 1×10⁷ to 1×10¹ copies/µl were assayed to evaluate the reproducibility, and three independent HTNV cRNA dilutions were tested in a single run to evaluate the intra-assay variance. The inter-assay variance was measured by testing each dilution in three independent runs, and the mean, SD and CV values were separately calculated for each HTNV cRNA dilution.