Simultaneous rapid detection of Hantaan virus and Seoul virus using RT-LAMP in rats

Ethics statement

This research was approved by the Experimental Animal Ethics Committee of Jinzhou Medical University (Approval: 20150918).

Samples

The purified HTNV strain RH153 and SEOV strain RH164 were gifts from Professor Liu Xuesheng of Liaoning Province center for disease control and prevention (CDC), and stored at −80 °C; lymphocytic choriomeningitis virus (LCMV), mouse poxvirus (MPV), rotavirus (RV), and mouse hepatitis virus (MHV)-infected rat lungs were donated by Professor Zhengming He of the National Institutes for Food and Drug Control (China), and influenza virus (IV), Coxsackievirus A16 (CA16), and lungs of healthy rat were preserved in Jinzhou (CDC), and stored in a liquid nitrogen; The clinical lung sample were collected from the captured rat in the Heishan, Guta, Beizhen, and Taihe of Jinzhou during 2008–2013, which were trapped with rat traps or baited cages in the tussocks, canals and fields, then stored in a liquid nitrogen until further processing.

Primers design

By comparison with HTNV and SEOV stains of China, S gene of SEOV strain Rn-HD164 (GenBank: GQ279392.1) was used as the reference sequence to design the primers of RT-LAMP (Figs. S1, S2 and Word S1). In order to ensure the specificity of the assay, six primers were designed to identify six regions of the S target gene, and five set RT-LAMP primers for HTNV and SEOV were designed based on the S gene sequence of Rn-HD164 strain using the website (http://primerexplorer.jp/e/).

Each set of primers contained two inner primers (FIP and BIP), two outer primers (F3 and B3), and two loop primers (LB and LF). The primer sequences used in the RT-PCR assays have been referred to National Health and Family Planning Commission of the People’s Republic of China. Primers were synthesized by Shanghai Sangon Biotech (Shanghai, China), and the sequences and positions of the RT-LAMP and RT-PCR primers are shown in Table 1.

RNA extraction

Lung tissue (about four mm³) from sample rats were obstained antiseptically in the Class II biological safety cabinet, then total RNA was extracted using the Qiagen RNAmini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s directions. cDNA was synthesized with Oligo dT-Adaptor Primer and first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The contents and volume of reaction mixture were as follows: MgCl₂ two μL, 10×RT Buffer one μL, RNase Free dH₂O 3.75 μL, dNTP Mixture (10 mM) one μL, RNase Inhibitor 0.25 μL, AMV Reverse Transcriptase 0.5 μL, Oligo dT-Adaptor Primer 0.5 μL which made up to 10 μL. The reaction conditions were as follows: 30 °C for 10 min, 42 °C for 30 min, 50 °C for 30 min, 95 °C for 15 min, and 5 °C for 5 min. The cDNA was stored at −20 °C. Viral RNA was extracted using the TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions.

RT-LAMP assays

The LAMP reaction was performed in 25-μL volume which contained 20 mM Tris-HCl (pH 8.8) (Shanghai Bioscience Biological Technology Company, Shanghai, China), 10 mM KCl and 10 mM (NH₄)₂SO₄ (Sinopharm Chemical Reagent Co., Shanghai, China), 0.1% Triton X-100 (Solebo Biotechnology Corporation, Beijing, China), 0.8M lysine (Sigma-Aldrich, St. Louis, MO, USA), eight mM MgSO₄ (Sinopharm Chemical Reagent Co., Shanghai, China), 1.4 mM dNTP, eight U of Bst DNA polymerase and reverse transcriptase, 40 pmol of each primer FIP and BIP, five pmol of each primer F3 and B3, and 20 pmol of each primer LB and LF. The reaction was performed at 65 °C for 60 min, and double-distilled water was used as a negative control.

Loop-mediated isothermal amplification products were analyzed by two independent methods, either sample turbidity or fluorescence. The LA-320 turbidimeter (Eiken Chemical Co., Ltd, Tokyo, Japan) was used to monitor the turbidity of RT-LAMP products in real time (measured at 6-s intervals on 650 nm wave length), and the samples were considered positive if the value of sample turbidity exceeded 0.1. The turbidity changes arose from the presence of LAMP reaction by-product a white precipitate (Mg₂P₂O₇). The second method used visual inspection to assess color changes in the presence of the fluorescent metal ion indicator calcein/Mn²⁺ complex, and the samples that turned from orange to green on reaction completion, while no color change samples, were considered negative.

Optimization of primers and temperature for RT-LAMP assay

To determine the optimal primer for RT-LAMP assay for HTNV and SEOV detection, five primer sets were detected in the same reaction condition using real-time turbidimeter LA-320 and their turbidity curves were draw at 650 nm according to the amplified results. Then the optimal primers were used to initiate RT-LAMP reaction at different temperatures ranging from 59 to 66 °C with a gradient of 1 °C.

RT-PCR assays

Reverse transcriptase polymerase chain reaction was conducted in accordance with the manufacturer’s protocol. Briefly, five μL of RNA sample was added to a 20 μL reaction mixture containing 100 ng Oligo dT (15mer), 1X AMV/Tfl buffer, one mmol/L MgSO₄, 10 mmol/L DTT, 200 μmol/L of each dNTP, 300 nM each specific primer, 2.5 units each of AMV Reverse Transcriptase and Tfl DNA Polymerase and 20 U RNasin. RT-PCR assays was followed uninterrupted thermal cycling programs consisting of 45 min at 42 °C, 4 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C and a final elongation step of 10 min at 72 °C. RT-PCR products were electrophoresed in 1% agarose gel and visualized using UV light after ethidium bromide staining.

Sensitivity comparison of RT-LAMP and RT-PCR

For comparison of sensitivity between RT-LAMP assay and RT-PCR, the positive controls (cRNA templates of the purified HTNV strain RH153 and SEOV strain RH164) were detected on the same samples. After the cRNA template concentration of the purified HTNV and SEOV was measured using the NanoDrop One microvolume UV–Vis spectrophotometer (ThermoFisher, Waltham, MA, USA), the viral copy numbers were calculated from the full length genome using the formula (copies/μL = ng/μL × Avogadro constant/1 bases of average molecular mass × genome length), then seven assay standards were prepared by 10-fold serial dilutions containing 10⁵–10⁻¹ copies/μL, respectively. The cRNA of the healthy rat lung tissue was used as the negative control. The colorimetric method (the addition of calcein) and the turbidity method (determination of turbidity) were both used to monitor the reaction of LAMP. At the same time, the RT-PCR products were electrophoresed in 1% agarose gel and visualized using UV light.

Specificity test of RT-LAMP

The specificity of RT-LAMP was assessed by testing cDNAs generated from CA16, IV, LCMV, MPV, RV, MHV, lungs of healthy rats and HTNV, SEOV positive samples. Distilled water was used as negative control.

Examination of clinical samples

A total of 23 rat lung samples were randomly selected from those collected between 2008 and 2013. All samples were collected according to the national guideline for prevention and treatment of epidemic hemorrhagic fever which states that night trapping method was used to seize rats randomly in the countryside and residential area (Zhang et al., 2004). Cordotomy was used to kill all the rats. Each of them was tested with RT-PCR, IFT, and RT-LAMP.

Optimization of primers

To determine the optimal primers for HTNV and SEOV, five primers were detected in the same reaction condition using real-time turbidimeter L320C and their turbidity curves were screened at 650 nm according to the amplified results. As illustrated in Fig. 1, HS-72 was selected as optimal primers (Table 1).

Determination of optimal reaction temperature

The optimal primers HS-72 were used to initiate RT-LAMP reaction at different temperatures ranging from 59 to 66 °C with a gradient of 1 °C. As shown in Fig. 4, RT-LAMP was first initiated at 64, 65, and 66 °C. However, the higher temperature could have an effect on the activity of Bst DNA polymerase and the reaction time was longer with temperature increasing, so 65 °C was selected as the optimal temperature.

Specificity test

HS-72 was used with all the provided samples in subsequent reactions. HS-72 displayed high specificity and could be used for simultaneous detection of HTNV and SEOV. Only HTNV and SEOV were detected with HS-72 and none of the samples with other viruses were diagnosed as positive, suggesting a good specificity (Figs. 2A and 2B).

Sensitivity test and comparison with PCR

For templates at concentrations from 1 × 10⁵ to 1 × 10⁻¹ copies/L, RT-LAMP assays were carried out by the LA-320C turbidimeter. As illustrated in Figs. 3A–3D, both the turbidity and color detection limit of the RT-LAMP assay was up 10 copies/μL for HTNV and SEOV. Comparison to the RT-LAMP assay, the RT-PCR limits of detection for HTNV and SEOV were 10² and 10³ copies/L, respectively (Figs. 3E and 3F).

Test results of clinical samples

The 23 clinical samples were detected to determine the feasibility of the RT-LAMP assay, and result have shown in Figs. 5A and 5B. At the same time, all the samples were subjected to conventional RT-PCR and IFT. In the result, the RT-LAMP method showed that five samples were determined as the positive of SEOV and HTNV, and four positive specimen in RT-PCR and only three in the IFT assay, which was superior to RT-PCR and IFT methods (Table 2). From shown in Table 2, all three methods yielded positive results for the JZHS-5, LZLH-4, JZGT-1 samples, and JZGT-2 sample wasn’t detected using RT-PCR, but the detection of RT-LAMP were positive.