Comparison of reverse-transcription loop-mediated isothermal amplification and reverse-transcription polymerase chain reaction for grass carp reovirus

Grass carp reovirus (GCRV) has been assigned to a newly established Aquareovirus genus in the family of Reoviridae which leads to haemorrhagic disease and extremely high mortality rate in grass carp. In this study, comparison was made between the novel one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) and the reverse transcription polymerase chain reaction (RT-PCR) for detection of grass carp reovirus. The result indicated that RT-LAMP had × 10 higher sensitivity comparable to RT-PCR. The specificity of the two methods for GCRV detection were both developed successfully by other three aquatic viruses. In the field trial, both RT-PCR and RT-LAMP methods were applied to detect the samples from different infected organs and tissues. The result demonstrated that RT-LAMP had a high accuracy to confirm the diagnosis as well as the RT-PCR. This study showed that the RT-LAMP, compared to the RT-PCR, was simple, time-saving, convenient, but required specificity primers and possibly generated false positive product. Its products, unlike RT-PCR, could not be direcly used in further molecular research after purification. Thus RT-LAMP might be an optimal diagnostic method for rapid and preliminary diagnosis of GCRV infection in resource-limited setting situation. Comparison, RT-PCR, one-step RT-LAMP, GCRV Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae, mainly infecting grass carp (Ctenopharyngodon idella) (Ma et al. 2011), which is one of the most promising species for aquaculture in China (Su et al. 2009; Ma et al. 2011). Because of the rapid spread and difficulty of treatment in freshwater aquaculture, GCRV causes a high mortality rate (about 80%) and enormous economic loss every year (Zhang et al. 2012). In terms of controlling the spread of infection, several immune diagnostic methods have been developed. The infection continues to occur. Therefore, earlier detection and effective prevention of GCRV play a crucial role in preventing and controlling its outbreaks. So many current methods have been used to diagnose the GCRV, including antigenrelated serological reactions, genome-related nucleic acid hybridization (Rangel et al. 1999), reverse transcription-polymerase chain reaction (RT-PCR) techniques and realtime RT-PCR (Zhou et al. 2011; Zhang et al. 2012). Real-time PCR has been considered to be the “golden standard” for accurate, sensitive, and rapid measurement for target sequence amplification (Watzinger et al. 2006). It has been broadly used for detecting and monitoring infections and aquatic viral load; however, these techniques require centralized laboratory facilities and clinical specimen submissions that might delay disease diagnosis. In recent years, an elegant and innovative technique called the loop-mediated isothermal amplification (LAMP) method has been successfully applied for detecting many kinds of aquatic bacteria and virues, such as Flavobacterium psychrophilum (Fujiwara and Eguchi 2009), Vibrio alginolyticus (Cai et al. 2010), haematopoietic necrosis virus (IHNV) (Gunimaladevi et al. 2005), haemorrhagic septicaemia virus (VHS) (Soliman ACTA VET. BRNO 2015, 84: 215–223; doi:10.2754/avb201584030215 Address for correspondence: Kai-Yu Wang Department of Basic Veterinary Medicine College of Veterinary Medicine Sichuan Agricultural University, Sichuan, China Phone: +86-0835-2885753 Fax: +86-0835-2885302 E-mail: [email protected] http://actavet.vfu.cz/ and El-Matbouli 2006), yellow head virus (YHV) (Mekata et al. 2006), spring viraemia of carp virus (SVCV) (Shivappa et al. 2008), and white spot syndrome virus (Jaroenram et al. 2009). The RT-LAMP, utilizing two sets of primers including two inner primers and two outer primers, are specific for six independent regions of the target sequence. A large amount of magnesium pyrophosphate is produced during the reaction, so the amplified products can be easily observed by the naked eye or judged visually by the colour change after the addition of nucleic acid stain (SYBR Green I) (Notomi et al. 2000). The purpose of this study was to establish an RT-LAMP method for GCRV that could be used as a diagnostic method in laboratories with limited equipment, especially under field conditions. Comparing the RT-LAMP with RT-PCR, the specificity and sensitivity of the two methods were assessed, and the applicability as diagnostic test was evaluated. Materials and Methods RNA extraction and viral strains Ribonucleic acid was extracted from the liver and spleen of the clinical grass carp samples, which were collected from Meishan farms in the Sichuan Province, using a commercial RNA Rapid Extraction Kit (Bio Teke, Beijing, China). Spring viraemia of carp virus (SVCV), haemorrhagic septicaemia virus (VHS) and koi herpesvirus (KHV) were provided by Dr. Q. Wang. (Department of Basic Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, Sichuan, China). The cDNA was obtained by the PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA, Dalian, China). Primers design Primers design was based on the target GCRV sequences from GenBank, aligned using ClustalW to find a well-conserved region within the gene (accession no. VP7/AF236688). A set of five primers was designed for RTLAMP assay using the online LAMP primer design software Primer Explorer V4 and RT-PCR primers displayed the location of oligonucleotide primers in the target sequence (Table 1). RT-PCR and optimization of reaction condition The RT-PCR was performed in a total volume of 25 μl containing the cDNA template (2 μg), primers F and R (2 μM/ each), 2.5 mM of dNTPs (Takara Biotechnology, Dalian, China), 25 mM MgCl2, Taq DNA polymerase (5 U/μl) (Takara Biotechnology, Dalian, China), 10 × PCR buffer consisting of 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2 SO4, 2 mM MgCl2, 0.1% Triton X-100 (HaiGene, Harbin, China), and ddH2O. The RT-PCR reaction mixture was incubated in an initial denaturation at 94 °C for 5 min; followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 53 °C for 45 s, extension at 72 °C for 45 s; finally extension at 72 °C for 10 min. The amplicon was analyzed in 1% agarose gel electrophoresis stained with ethidium bromide. In order to optimise the assay, the reaction conditions, including the dosage of template, primers, dNTPs, Taq DNA polymerase, MgCl2 and annealing temperature, were evaluated. 216 GCRV: grass carp reovirus; RT-PCR: reverse-transcription polymerase chain reaction; RT-LAMP: reversetranscription loop-mediated isothermal amplification Method Primer Type Sequence (5’to 3’) RT-PCR F Forward primer 5’-TCAAGACTCCCACGCTTGTTC-3’ R Reverse primer 5’-TGCGTATCGTCCAACGGTTT-3’ RT-LAMP F3 Forward outer primer 5’-TGGATGTGAAAGCTGGGTTC-3’ B3 Backward outer primer 5’-GGGATGCTCGTTAGGCAATT-3’ FIP Forward inner primer 5’-GGTGCGAAACGGAAGTTCGACGCC CCAACTGCCGATGAAAC-3’ BIP Backward inner primer 5’-ACGATTCCTCTGCTACCGCTTG AAAGGATCGGGAGGTGGAT-3’ LB Backward loop primer 5’ATCACTGCCAGGCCGGTCA-3’ Table 1. Details of RT-PCR and RT-LAMP primers designed for detection of VP7 gene sequence of GCRV. Genome position depending on GCRV VP7 (225-569) gene sequence (GenBank accession no. AF236688). The primers of VP7-F and VP7-R were for RT-PCR primers, VP7-F3, VP7-B3, VP7-FIP, VP7-BIP, and VP7-LB were applied in RT-LAMP. One-step RT-LAMP assay and optimization of reaction condition The RT-LAMP reaction mixture (total volume 25 μl) contained 2 μl of target RNA, 0.125 U of AMV reverse transcriptase (Promega, Madison, WI), F3 and B3 (5 pmol/each), FIP and BIP (50 pmol/each), 25 pmol of primer LB, 2.5 mM of dNTP mix (Takara Biotechnology, Dalian, China), 25 mM MgCl2, 8 M betaine (Sigma, St. Louis, USA), 10 × Bst DNA polymerase buffer, 8 U of Bst DNA polymerase (Takara Biotechnology, Dalian, China), and ddH2O. Reaction was carried out at 63 °C for 60 min and heated at 80 °C for 5 min. The following indicators of the optimal RT-LAMP amplification were evaluated: ratios of outer and inner primers (1:1 to 1:16); Mg2+ concentration (2–12 mM) (including the 2 mM Mg2+ in the buffer); dNTPs concentration (0–2.0 mM); amount of template; amount of Bst DNA polymerase (0.4–8 U); temperature (59–66 °C), and RT-LAMP reaction time (10–60 min). All of the precautions of cross-contamination were observed. Each amplicon (5 μl well) was electrophoresed in a 1.5% agarose gel electrophoresis stained with ethidium bromide (Li and Ren 2011). Comparison of specificity and sensitivity of virus detection by RT-LAMP and RT-PCR In order to determine the specificity of the RT-LAMP and RT-PCR assays, the specificity assay was evaluated by using the other three fish viruses, including spring viraemia of carp virus (SVCV), haemorrhagic septicaemia virus (VHS) and koi herpesvirus (KHV). To determine the sensitivity, the template was serially diluted in tenfold decrements down to 10-4 in ddH2O and was used in both assays independently. Field trial To evaluate the reliability of the RT-LAMP and RT-PCR assays as on-site diagnosis system, 48 clinical samples were tested to determine the feasibility, which were collected from grass carps suspected of GCRV infection 217 Fig. 1. Optimization of the RT-PCR reaction for GCRV. (A) The effect of annealing temperature: lanes 1–6 (50, 50.5, 51.5, 52, 53.5, and 54.5 °C, respectively). (B) The effect of MgCl2 (2 mM): lanes 1–7 (0, 2, 4, 6, 8, 10 and 12 mM, respectively). (C) The effect of dNTPs (1.4 mM): lanes 1–6 (1, 2, 4, 6, 8 and 10 mM, respectively). (D) Each of the forward and reverse primers (2 μM/ each): lanes 1–6 (5, 4, 3, 2, 1, and 0.5, respectively). (E) The effect of Taq polymerase (5 U/μl): lanes 1–6 (0.6, 0.5, 0.4, 0.3, 0.2, and 0.1 μl, respectively). M: DL 2000 marker. M 1 2 3 4 5 6