Virus and samples
CSFV (shimen strain), Porcine epidemic diarrhea virus (PEDV, CV777 strain), Porcine parvovirus (PPV, GDQY/2010/01 strain), Pseudorabies virus (PRV, Ea strain), Porcine reproductive and respiratory syndrome virus (PRRSV, 08 − 2 strain), Japanese encephalitis virus (JEV, JEV/sw/GD/2009 strain), and Porcine circovirus type 2 (PCV2, LG strain) were preserved in our laboratory. A total of twelve samples including inguinal lymph nodes, spleen, thymus, and tonsil from three pigs experimentally infected with CSFV (shimen strain) in our previous work were preserved in our laboratory. These samples were tested for CSFV by RT-RPA-LFD assay and RT-PCR.
Isolation of viral DNA/RNA and synthesis of cDNA
Viral DNA/RNA was extracted from samples using a Viral DNA/RNA Extraction Kit (OMEGA Bio-Tek, USA) according to the manufacturer’s instructions. The viral cDNA was synthesized using M-MLV reverse transcriptase (TAKARA Biotechnology, Dalian, China) according to the manufacturer’s instructions. All viral DNA, RNA, and cDNA were stored at -80 °C for further employment.
CSFV RT-PCR assay
The cDNA of CSFV was synthesized as above and used for PCR amplification. The primers used for PCR were CSFV-f (5’-CTAGCCATGCCCWYAGTAGG-3’) and CSFV-r (5’-CAGCTTCARYGTTGATTGT-3’). A 421 bp fragment within 5’-UTR region of the CSFV genome was amplified. The amplification was performed in a 25 µL volume containing 2 × Taq PCR StarMix (12.5 µL), ddH2O (8.5µL), the forward and reverse primers (10 µM, 1 µL each) and cDNA template (2 µL). PCR amplification was performed as follows: 5 min of initial denaturation at 94 oC, 35 cycles of 30 s of denaturing at 94 oC, 30 s of annealing at 55 oC, and 30 s of extension at 72 oC. The 10 min of extension at 72 oC was added after the 35-cycle amplification. The PCR products were detected with 1% agarose gels.
CSFV RT-RPA-LFD assay
The cDNA of CSFV was synthesized as above and used for RPA assay. RPA primers and probes were designed according to the parameter requirements of the TwistAmp™ nfo RPA kit manual (TwistDx™ Limited, Cambridge, UK) based on the CSFV genome sequence (GenBank Accession number: HQ380231) using Primer Premier 5.0. The reverse primer was labeled with a biotin at the 5’ end and the nfo-probe carried a fluorescein FAM at the 5’ end, an internal tetrahydrofuran residue (THF) at 30 bp from the 5’ end, and a C3 spacer at the 3’ end (Table 1). All the primers and probes were synthesized by Sangon Biotech (Shanghai, China). The RPA assay was performed in a 50 µL final reaction volume according to the instructions of the TwistAmp™ RPA nfo kit (TwistDx™ Limited, Cambridge, UK). In brief, the rehydration solution contained 4 µL cDNA template, 2.1 µL of forward primer (10 µM), 2.1 µL of reverse primer (10 µM), 0.6 µL of the probe (10 µM), 9.2 µL of ddH2O, and 29.5 µL rehydration buffer (TwistDx™ Limited, Cambridge, UK). The dried enzyme pellet in a 0.2 mL tube strip provided by the TwistAmp™ RPA nfo kit was resuspended with 47.5 µL of the rehydration solution, and then the reaction was initiated by adding 2.5 µL of magnesium acetate (280 mM). Experiments were performed to optimize the RPA assay by testing different incubation time and amplification temperature. The RPA result was directly determined using lateral flow dipstick (LFD) visualization strip cassette (Ustar Biotech, Hangzhou, China). The manipulation procedure of the LFD visualization strip cassette was shown in Fig. 5A. Firstly, the reaction tube was pushed transversely into the amplicon cartridge. Next, the cartridge was closed and inserted into the detection chamber. Then, the handle of the detection chamber was pressed to be sealed. Once the handle being closed, both the reaction tube and the bulb containing the running buffer were cut by the razor blade and the amplicon products mixed with the running buffer and flowed to the DNA detection strip which was implanted into the cassette through a fiberglass paper. One to two minutes after the handle being closed, the result could be determined based on the displayed bands from the detection window of the chamber. As shown in Fig. 5B, the red-purple bands both at T line and C line could be detected in the positive sample while only one band (C line) appeared for a negative sample.
The specificity and sensitivity of CSFV RT-RPA-LFD assay
The specificity of CSFV RT-RPA-LFD assay was evaluated by detecting different species of viruses including CSFV, PEDV, PPV, PRV, PRRSV, PCV2, and JEV. The sensitivity of the RT-RPA-LFD assay and RT-PCR were compared by detecting ten-fold serial dilutions of CSFV cDNA.