Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Rapid and sensitive detection of Senecavirus A by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick method

  • Jinhui Li,

    Roles Data curation, Formal analysis, Project administration, Writing – original draft, Writing – review & editing

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Weifang Liang,

    Roles Software, Validation, Writing – review & editing

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Shuaifei Xu,

    Roles Conceptualization, Supervision, Validation, Visualization

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Jian Shi,

    Roles Conceptualization, Data curation, Formal analysis

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Xia Zhou,

    Roles Funding acquisition, Investigation, Methodology

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Bowen Liu,

    Roles Project administration, Resources, Software

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Li Yu,

    Roles Software, Supervision, Validation

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Jingfeng Xiong,

    Roles Supervision, Validation, Visualization

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Guangbin Si,

    Roles Project administration, Resources, Software

    Affiliation College of Veterinary Medicine, South China Agricultural University, Guangzhou, China

  • Dongsheng He

    Roles Conceptualization, Data curation, Visualization, Writing – original draft, Writing – review & editing

    dhe@scau.edu.cn

    Affiliations College of Veterinary Medicine, South China Agricultural University, Guangzhou, China, Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, Guangzhou, China, Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, Guangzhou, China

Rapid and sensitive detection of Senecavirus A by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick method

  • Jinhui Li, 
  • Weifang Liang, 
  • Shuaifei Xu, 
  • Jian Shi, 
  • Xia Zhou, 
  • Bowen Liu, 
  • Li Yu, 
  • Jingfeng Xiong, 
  • Guangbin Si, 
  • Dongsheng He
PLOS
x

Abstract

Senecavirus A (SVA) is a critical pathogen causing vesicular lesions in sows and acute death of newborn piglets, resulting in very large economic losses in the pig industry. To restrict the transmission of SVA, an establishment of an effective diagnostic method is crucial for the prevention and control of the disease. However, traditional detection methods often have many drawbacks. In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was combined with a lateral flow dipstick (LFD) to detect SVA. The resulting RT-LAMP-LFD assay was performed at 60°C for 50 min and then directly judged on an LFD visualization strip. This method shows high specificity and sensitivity to SVA. The detection limit of RT-LAMP was 4.56x10-8 ng/μL RNA, approximately 11 copies/μL RNA, and it was 10 times more sensitive than RT-PCR. This detection method’s positive rate for clinical samples is comparable to that of RT-PCR. This method is time saving and highly efficient and is thus expected to be used to diagnose SVA infections in this field.

Introduction

Senecavirus A (SVA), formerly known as Seneca Valley virus (SVV)[1], is the only recognized species of the genus Senecavirus within the family Picornaviridae[2], and it was first isolated in 2002[3]. SVA is clinically characterized by vesicular ulcers in the coronary arteries or nostrils[4]. Therefore, it is difficult to differentiate from foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS) in clinical symptoms[5]. SVA is a small, nonenveloped RNA virus with a single sense strand 27–30 nm in diameter. The viral genome is approximately 7.3 kb in length and consists of a 5’ noncoding region (UTR), a 3’ noncoding region (UTR) and a large open reading frame[3]. The only open reading frame encodes a polymeric precursor protein including an L leader protein, a P1 region structural protein, and the P2 and P3 region nonstructural proteins, which is very similar to other members of the small RNA virus family (Picornaviridae)[2, 3]. From the 5' to 3' direction, the genomic structure L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D is the same as that of the picornavirus L-4-3-4 genome structure[3].

SVA is clinically difficult to distinguish from other classic viral vesicular diseases[6], which increases the difficulty of clinical diagnosis. Therefore, it is necessary to establish a series of laboratory diagnostic methods for the diagnosis and identification of vesicular diseases. Reverse transcription droplet digital PCR assay (RT-ddPCR) [7], Indirect immunofluorescence assay (IFA)[8], reverse transcription polymerase chain reaction (RT-PCR)[9, 10], reverse transcription quantitative PCR (qRT-PCR)[11], novel RNA-based in situ hybridization[12] and enzyme-linked immunosorbent assays (ELISAs)[13] have also been applied to detect SVA. These methods show high specificity and sensitivity but place high demands on experimental instruments and the skills of research personnel. Therefore, the above methods are not suitable for laboratories with poor clinical and experimental equipment.

Loop-mediated isothermal amplification (LAMP) is a new PCR-based molecular amplification method invented by Notomi in 2000[14]. It can efficiently, rapidly and specifically amplify the target sequence under isothermal conditions and relies on primers that have the ability to recognize six specific regions on the target sequence, a Bst DNA polymerase with a helicase function and LAMP of the target sequence[1417]. LAMP techniques have high specificity, sensitivity, and stability, and studies have shown that LAMP has similar sensitivity as ordinary PCR[1820]. LAMP can detect RNA templates by using reverse transcriptase[14, 21], and the RT-LAMP method has been applied to the detection of multiple viruses[18, 2224]. Amplification by this method can occur in a common water bath, and the amplified product can be visually identified by adding a fluorescent dye[25]. The method is suitable for the detection of a primary layer and the clinical site.

The lateral flow dipstick (LFD) method utilizes a biotin-labeled LAMP amplification product to specifically hybridize to a probe labeled with fluorescein isothiocyanate (FITC), which thereby binds to a colloidal gold-labeled anti-FITC antibody to form a ternary complex that is bound at a biotin-containing detection line, while unhybridized FITC-labeled probe forms a biotin-free binary complex and is bound at an anti-FITC quality control line[26]. Recently, an RT-LAMP-LFD assay has been successfully used for the detection of a variety of viral pathogens[2730]. The method has the advantages of high specificity, high sensitivity, good stability and repeatability. In addition, the RT-LAMP-LFD assay avoids false positives caused by agarose gel electrophoresis or fluorescent dye staining[31].

In this study, LAMP primers were designed to target the conserved regions of the SVA 3D gene sequence. An SVA RT-LAMP method was established. We combined RT-LAMP with a LFD visualization strip to develop an RT-LAMP-LFD assay that facilitates the diagnosis of SVA. The method has excellent specificity, high sensitivity, and good stability and repeatability and provides a reliable method for preliminary screening and detection of SVA in ordinary laboratories and at sites in the field.

Materials and methods

Viruses

Senecavirus A (SVA; HN16 strain, Accession number: MF893200), porcine deltacoronavirus (PDCoV; PDCoV/CHGD/2016 strain, Accession number: MH715491), porcine epidemic diarrhea virus (PEDV; CH/GDGZ/2012 strain, Accession number: KF384500), porcine transmissible gastroenteritis virus (TGEV; CN12 strain, Accession number: KX058075), and porcine reproductive and respiratory syndrome virus (PRRSV; JXA1 strain, Accession number: EF112445) were obtained from the virus repository of the College of Veterinary Medicine of South China Agricultural University. A foot-and-mouth disease virus (FMDV) vaccine strain and classical swine fever virus (CSFV) standard positive serum were purchased from the Beijing Century Yuanheng Animal Epidemic Prevention Technology Co., Ltd. The whole genome of swine vesicular disease virus (SVDV; HK'70 strain, Accession number: AY429470) and vesicular stomatitis virus (VSV; Arizona/5481555/2015 strain, Accession number: KT429217) was synthesized by Beijing Qingke Biotechnology Co., Ltd.

Clinical sample collection and testing

From September to December 2017, 33 clinical serum samples were collected from different suspected pig herds in Guangzhou, Guangdong Province. Samples were collected from weaned piglets and sows. Blood samples were collected from the ear-rim auricular vein using a 5ml disposable sterile syringe. Blood sample of 4 ml was collected from each suspected infected pig. The collected blood was placed in a 5 ml sterile centrifuge tube and allowed to stand at room temperature for 30 min. Serum samples were obtained by centrifugation at 1200 g for 10 min. All serum samples were shipped at 0°C and stored at -80°C until total RNA was isolated. The sampling procedure was approved by the Animal Ethics Committee of Guangdong Province, China. The sampling process was assisted by local authorities and veterinarians. Samples of the disease were obtained after veterinary pathological examination.

Nucleic acid extraction

Viral RNA of SVA (HN16 strain), PDCoV (PDCoV/CHGD/2016 strain), PEDV (CH/GDGZ/2012), TGEV (CN12 strain), and PRRSV (YA strain) were extracted from 300 μL of infected cell culture supernatant. Meanwhile, viral RNA of the above viruses, the FMDV vaccine strain and classical swine fever virus (i.e., CSFV) standard positive serum were extracted using TRIzol reagent (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions. In addition, 300 μL of each clinical serum sample was subjected to RNA extraction using TRIzol reagent. The extracted RNA was resuspended in 50 μL of RNase-free water (Gibco BRL, USA) and stored at -80° C until further testing. Positive plasmids of SVDV and VSV were extracted by Plasmid Mini Kit I (200) (Omega Bio-tek, Norcross, GA, USA).

Primers

Thirty-one full-length sequences of SVA were obtained from GenBank(GenBank Accession numbers: MF893200, MK256736, NC-011349, KR063107, KT321458, KT757282, KU359210, KU359214, KU954090, KX223836, KX857728, KY038016, KY419132, KY486163, KY618837, KY747510, KY747511, KY747512, MF416220, MF967574, MG428680, MG428681, MG428682, MG428683, MG428684, MG428685, MG765550, MG765552, MH316114, MH704432) and aligned by MegAlign (Larsergene, version 7.0), and it was found that its 3D region was more conservative (S1 Fig). LAMP primers were designed based on the conserved regions of the SVA 3D gene sequence. RT-LAMP primer-assisted design software Primer Explorer V5 (http://primerexplorer.jp/e/) was used to design the RT-LAMP primers of the SVA gene based on the determined conserved nucleotide sequence (HN16 strain, GenBank Accession number MF893200). The RT-LAMP primer sets for detecting SVA includes a forward primer (SVA-FIP), a reverse internal primer (SVA-BIP), a forward external primer (SVA-F3), a reverse external primer (SVA-B3), a forward loop primer (SVA-LF) and a reverse loop primer (SVA-LB). The FIP primer was labeled with biotin at the 5' end, and the BIP primer was labeled with FITC at the 5' end (Table 1). In addition, the conserved region of the SVA VP1 gene sequence was used in the design of common RT-PCR detection primers (Table 2). The synthesis and labeling of all primers was done by the BGI TECH SOLUTIONS (BEIJING LIUHE Co., Ltd, Beijing, China) (Fig 1).

thumbnail
Fig 1. Schematic showing location of RT-LAMP primer binding sites within SVA 3D gene.

SVA RT-LAMP primer binding sites. The assay spans nucleotides 6615–6908 with reference to the 3D gene sequence of the SVA virus strain SVA-HN16.

https://doi.org/10.1371/journal.pone.0216245.g001

Optimization of reaction conditions for loop-mediated isothermal amplification

Establishment of initial RT-LAMP-LFD detection method.

The RT-LAMP assay was carried out in a 25 μL reaction mixture system containing 10×ThermoPol Buffer (New England Bio-labs, USA), 2.5 mM dNTPs (TaKaRa Biotechnology, Dalian, China), 10 μM SVA-FIP and SVA-BIP, 10 μM SVA-LF and SVA-LB, 10 μM SVA-F3 and SVA-B3, 100 mM MgSO4 (New England Bio-labs, USA), 8 U/μL Bst DNA polymerase (8U, New England Bio-labs, USA), 5 U/μL AMV reverse transcriptase (5 U, TaKaRa Biotechnology, Dalian, China) and 3 μL (1.368ng) of RNA was used as a template or 3 μL of ddH2O as a template for a negative control. The amplification was incubated for 60 min in a constant temperature water bath at 60°C, then the reaction was terminated by raising the temperature to 80°C for 4 min. Subsequently, the reaction product in the reaction tube was used for LFD (Ustar Biotech, Hangzhou, China) visual test strip detection. The tested samples included a negative control without a template. After 10–15 min, the results were observed by eye and recorded. If both the T line and the C line appeared, the result was a positive amplification. If only the C line was displayed, the result was a negative amplification. When both the T line and C line were not visible, the LFD was considered invalid. The RT-LAMP products were also routinely analyzed by calcein (Beijing Solarbio Science & Technology Co., Ltd.) RT-LAMP assay and 1.2% agarose gel (Gibco BRL, USA) electrophoresis to verify the expected results of positive LAMP reaction products. Gel electrophoresis was performed in 1×TAE buffer (Gibco BRL, USA) at 120V for 35 minutes.

Optimization of SVA RT-LAMP reaction conditions.

To determine the optimal RT-LAMP reaction conditions, the temperature and time of the RT-LAM reaction were optimized. The RT-LAMP reaction was carried out with the 8 temperatures 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C and 64°C. Reaction products were analyzed by electrophoresis on 1.2% agarose gels, and the optimal reaction temperature was determined by the amplification effect. RT-LAMP reactions were carried out for the 8 times of 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min and 65 min. The reaction products were analyzed by electrophoresis on 1.2% agarose gels, and the optimal reaction time was determined by the amplification effect.

Optimization of RT-LAMP reaction system.

To determine the optimal RT-LAMP reaction conditions, the RT-LAM reaction system was optimized. The 6 Mg2+ concentrations 0 mM, 2 mM, 4 mM, 6 mM, 8 mM and 10 mM were used for the RT-LAMP reaction, and the remaining components were unchanged. The optimal Mg2+ concentration was determined according to the amplification effect. RT-LAMP reactions were carried out with the 8 dNTP concentrations 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM, 0.5 mM and 0.55 mM, and these experiments were carried out under optimized conditions and component concentrations. The optimal dNTP concentration was determined by the amplification effect. The 6 Bst DNA polymerase concentrations 0.08 U/μL, 0.16 U/μL, 0.24 U/μL, 0.32 U/μL, 0.4 U/μL and 0.48 U/μL were used for RT-LAMP reactions with optimized conditions and compositions, and these experiments were performed to determine the optimal Bst DNA Polymerase concentration based on the amplification effect. The 6 AMV concentrations 0.02 U/μL, 0.04 U/μL, 0.06 U/μL, 0.08 U/μL, 0.1 U/μL, and 0.12 U/μL were used for RT-LAMP reactions, and these experiments were performed with optimized conditions and compositions. The optimal AMV concentration is determined by the amplification effect. The ratio between the primers SVA-LF and SVA-LB was selected as a reference, and 8 concentration ratios of SVA-FIP and SVA-BIP (2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1) were tested with optimized conditions and compositions. The optimal concentration of SVA-FIP and SVA-BIP was determined by the amplification effect. In addition, the ratio of the primers SVA-FIP and SVA-BIP was picked as a reference, and 7 concentration ratios of SVA-LF and SVA-LB (0:1, 1:1, 2:1, 3:1, 4:1, 5: 1, 6:1) were tested with optimized conditions and compositions. Finally, the optimal concentration of SVA-LF and SVA-LB was determined by the amplification effect.

Specificity of SVA RT-LAMP-LFD

According to the optimized RT-LAMP reaction conditions, The viral RNA extracted from SVA (HN16 strain), PDCoV (PDCoV/CHGD/2016 strain), PEDV (CH/GDGZ/2012 strain), TGEV (CN12 strain), PRRSV (JXA1 strain), the FMDV vaccine strain), the CSFV standard positive serum and positive plasmids of SVDV and VSV were used as templates to test the specificity of the method. The reaction products were analyzed by LFD detection, calcein RT-LAMP assay and 1.2% agarose gel electrophoresis. Each test was repeated at least 3 times.

Sensitivity of SVA RT-LAMP-LFD

SVA standard template (45.6 ng/μl) were prepared from genomic total RNA extracted from 300 μl of SVA-infected PK-15 cell line culture supernatant as an initial reaction template. The initial reaction template was subjected to 10-fold serial dilution (10−1–10−9), and the reaction was carried out according to the optimized RT-LAMP reaction conditions. The reaction products were analyzed by LFD detection, calcein RT-LAMP assay and 1.2% agarose gel electrophoresis. Each test was repeated at least 3 times.

RT-PCR

TRIzol reagent was used for RNA extraction, and the reverse transcription reaction system was 20 μL in volume: 4 μL of 5×AMV Buffer (5U, TaKaRa Biotechnology, Dalian, China), 2 μL of dNTPs (2.5 mM), 0.5 μL of RNase inhibitor (40 U, TaKaRa Biotechnology, Dalian, China), 1 μL of random primer (50 μM, TaKaRa Biotechnology, Dalian, China), 1 μL of AMV, and 10 μL (4.56ng) of RNA, supplemented with ddH2O to 20 μL. The cDNA obtained by reaction at 42°C for 1 h was used as a template, and SVA-F and SVA-R were used as primers for PCR. The PCR system volume was 25 μL: 2.5 μL of 10 × PCR Buffer, 2 μL of dNTPs (2.5 mM), 0.5 μL of SVA-F (10 μM), 0.5 μL of SVA-R (10 μM), 0.25 μL of Ex-Taq polymerase (5 U, TaKaRa Biotechnology, Dalian, China), and 2 μL of template, supplemented with ddH2O to 25 μL. The PCR conditions were predenaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, 30 cycles; final extension at 72°C for 10 min. The reaction product was analyzed by 1.2% agarose gel.

Results

Establishment of initial RT-LAMP-LFD detection method

SVA HN16 strain RNA was used as a template for RT-LAMP reactions. The products were detected by 1.2% agarose gel electrophoresis, calcein assay and LFD assay to verify the feasibility of the RT-LAMP method. Agarose gel electrophoresis showed that the product resulting from the SVA-positive template had ladder-like bands and that the negative control had no bands (Fig 2A); the calcein method showed that the SVA-positive template product fluoresced green and that the negative control fluoresced orange (Fig 2B). The RT-LAMP-LFD results showed 2 red bands, one in the test line (T) and one in the quality control line (C), in the positive test group, while there was only one red strip (Fig 2C), in the quality control line (C), in the negative control group. The results show that the SVA RT-LAMP-LFD detection method is feasible.

thumbnail
Fig 2. Establishment of SVA RT-LAMP-LFD detection method.

A): RT-LAMP with gel electrophoresis detection: M: 2000 bp DNA marker, lane 1 for positive template (P), lane 2 for negative control (N); B): calcein RT-LAMP detection method: tube 1 for positive template (P), tube 2 for negative control (N); C): RT-LAMP-LFD detection method: strip 1 for positive template (P), strip 2 for negative control (N).

https://doi.org/10.1371/journal.pone.0216245.g002

Optimization of SVA RT-LAMP reaction conditions

In optimizing the SVA RT-LAMP reaction conditions, the RT-LAMP reaction was carried out at 8 different temperatures from 57–64°C, and the results of 1.2% agarose gel electrophoresis (Fig 3A) show that the enhanced band of lane 5 is the brightest, indicating that the optimum reaction temperature for the SVA RT-LAMP reaction is 61°C; the reaction time optimization results (Fig 3B) show that the brightness of the agarose gel electrophoresis band of the RT-LAMP reaction product was consistent after 50 min. Therefore, 50 min was selected as the optimal reaction time for the SVA RT-LAMP reaction. The optimization results for the reaction system are shown in Fig 4. The optimal reaction system of SVA RT-LAMP is 6 mM magnesium ions (Fig 4A), 0.5 mM dNTPs (Fig 4B). The optimal concentration of Bst DNA Polymerase was 0.32 U/μL (Fig 4C), the optimal AMV concentration was 0.06 U/μL (Fig 4D), and the optimal ratio of inner primer to external primer was 0.2 μM:0.12 μM (Fig 4E). The optimal ratio of loop primer to external primer was 0.04 μM:0.2 μM (Fig 4F).

thumbnail
Fig 3. Optimization of SVA RT-LAMP reaction conditions.

A): Reaction temperature optimization: M: 2000 bp DNA marker, lanes 1 to 8 represent 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, for a gradient with 8 temperatures; B): Reaction time optimization: M: 2000 bp DNA marker, lanes 1 to 8 respectively represent reaction times of 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, and 65 min, for a gradient with 8 reaction times.

https://doi.org/10.1371/journal.pone.0216245.g003

thumbnail
Fig 4. Optimization of SVA RT-LAMP reaction system.

M: 2000 bp DNA marker; A): Mg2+ concentration optimization, lanes 1 to 6 represent 0 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM, for 6 Mg2+ concentrations; B): dNTP concentration optimization, lanes 1 to 8 represent the 8 dNTP concentrations 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM, 0.5 mM, and 0.55 mM; C): Bst DNA polymerase concentration optimization, lanes 1 to 6 represent 0.08 U/μL, 0.16 U/μL, 0.24U/μL, 0.32U/μL, 0.4U/μL, and 0.48U/μL, for 6 Bst DNA Polymerase concentrations; D): AMV reverse transcriptase concentration optimization, lanes 1 to 6 represent the 6 AMV reverse transcriptase concentrations 0.02 U/μL, 0.04 U/μL, 0.06 U/μL, 0.08 U/μL, 0.1 U/μL, and 0.12 U/μL; E): optimization of internal primer to external primer ratio; in lanes 1 to 8, the ratio of the inner primer (IP) to the outer primer (OP) is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1; F): optimization of outer primer to external primer ratio; in lanes 1 to 7, the ratio of the loop primer (LP) to the outer primer (OP) is 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1.

https://doi.org/10.1371/journal.pone.0216245.g004

Specificity of SVA RT-LAMP-LFD detection method

To confirm the specificity of the SVA RT-LAMP-LFD detection method, the reaction was carried out using optimized reaction conditions. The viral RNA templates of SVA (HN16 strain), PDCoV (PDCoV/CHGD/2016 strain), PEDV (CH/GDGZ/2012 strain), TGEV (CN12 strain), PRRSV (JXA1 strain), the FMDV vaccine strain, the CSFV standard positive serum and positive plasmids of SVDV and VSV were separately detected by the RT-LAMP-LFD method and then analyzed by agarose gel electrophoresis and the calcein method. The results showed that SVA (HN16 strain) was positively amplified and the remaining pathogens were negatively amplified (Fig 5A–5C). The results indicated that the established SVA RT-LAMP-LFD detection method has good specificity.

thumbnail
Fig 5. Specificity of SVA RT-LAMP-LFD detection method.

M: 2000 bp DNA marker; lanes 1 to 10 are SVA, FMDV, SVDV, VSV, PDCoV, PEDV, TGEV, PRRSV, CSFV and negative control (N); A): specificity of RT-LAMP agarose gel electrophoresis assay; B): specificity of calcein RT-LAMP assay; and C): specificity of RT-LAMP-LFD assay.

https://doi.org/10.1371/journal.pone.0216245.g005

Sensitivity of SVA RT-LAMP-LFD detection method

To confirm the sensitivity of the SVA RT-LAMP-LFD detection method, the optimized reaction conditions were used for experiments, and a 10-fold gradient dilution of SVA HN16 genomic RNA was used as a template for the RT-LAMP reaction. The results showed that RT-LAMP agarose gel electrophoresis (Fig 6A), calcein RT-LAMP (Fig 6B) and RT-LAMP-LFD methods (Fig 6C) have the same sensitivity, and the lowest concentration of template detected was 4.5x10-8 ng/μL. However, this method is 10 times more sensitive than RT-PCR (4.5x10-7 ng/μL) (Fig 6D).

thumbnail
Fig 6. Sensitivity of SVA RT-LAMP-LFD detection method.

1: 4.56x10-1 ng/μL; 2: 4.56x10-2 ng/μL; 3: 4.56x10-3 ng/μL; 4: 4.56x10-4 ng/μL; 5: 4.56x10-5 ng/μL; 6: 4.56x10-6 ng/μL; 7: 4.56x10-7 ng/μL; 8: 4.56x10-8 ng/μL; and 9: 4.56x10-9 ng/μL; M: 2000 bp DNA marker; A): sensitivity of RT-LAMP agarose gel electrophoresis; B): sensitivity of calcein RT-LAMP under natural light; C): sensitivity of RT-LAMP-LFD; and D): sensitivity of RT-PCR.

https://doi.org/10.1371/journal.pone.0216245.g006

Clinical application of SVA RT-LAMP-LFD detection method

Thirty-three seral samples clinically suspected of SVA infection were tested by RT-PCR and RT-LAMP-LFD. The results (Table 3) showed that 18 samples were positively amplified by RT-PCR and that the positive rate was 67.4%. When tested by the RT-LAMP-LFD method, 18 samples were positively amplified, and the positive rate was 67.4%. The coincidence rate of the two methods was 100%. The SVA RT-LAMP-LFD assay has the same clinical detection rate as the conventional RT-PCR assay.

Discussion

In this study, we first described a rapid detection method of RT-LAMP-LFD for SVA, which combined RT-LAMP with a LFD visualization strip to specifically detect SVA. The optimum LAMP reaction conditions were 61°C for 50 minutes. The LAMP products labeled with the probe were then hybridized in a closed lateral flow dipstick, and the results were visually observed by the naked eyes after 10–15 min. The entire process can be completed in less than 1h and requires no special equipment.

Since the development of the LAMP method[14], a series of LAMP and RT-LAMP assays have been applied to detect various of different pathogens infecting animals and human beings. The LAMP method were also combined with other simple methods, thus generating a variety of determination methods for results, including agarose gel electrophoresis, usage of dyes such as hydroxynaphthol blue or calcein[32], monitoring of accumulation of insoluble magnesium pyrophosphate by a turbidity device[33], and fluorescence dye detection by a fluorescence monitoring equipment (eg, a real-time PCR machine). However, these methods have higher requirements for hardware devices, and it is not easy to perform rapid detection at sites in the field. At present, various detection methods have been developed for the detection of SVA, such as real-time PCR[34], novel RNA-based in situ hybridization[12], enzyme-linked immunosorbent assay (ELISA)[13], etc. In addition, the LAMP detection method for SVA has been reported[35], but the method in this study required the Genie II instrument, which had high technical requirements for the experimenter and has not been widely used in the detection at sites in the field.

In this study, the existing SVA RT-LAMP detection method was improved by labeling the FIP and BIP with biotin and fluorescein isothiocyanate respectively, and lateral flow strips coated with biotin antibodies and isothiocyanate fluorescein antibodies were used for rapid detection of LAMP products. Compared with that of RT-PCR, the detection time established by this method is much shorter, and the detection sensitivity is 10 times higher, while the detection sensitivity of this method is equivalent with that of qRT-PCR[11]. Compared with the calcein RT-LAMP method, the method established in this experiment eliminates human error caused by visual observation of color, and the test is completed in a closed container, thus eliminating false-positive amplification caused by additional contamination of fluorescent dye in the RT-LAMP product. In some studies, only one labeled primer was used in the LAMP reaction[36, 37], and another labeled probe was added to the LAMP products to form a double-labeled detectable product, which easily caused contamination of the LAMP product. In our study, the two internal primers were labeled with biotin isothiocyanate and fluorescein isothiocyanate respectively. The results obtained from this and other studies indicate that the use of both labeled primers has no adverse effect on the LAMP reaction and it also showed that the method has the advantages of reducing contamination and easier operation [3840].

In addition to the simple and rapid method of detecting RT-LAMP reaction products, this study also demonstrates that RT-LAMP amplification does not rely on strict constant temperature control and stringent time requirements. In temperature-optimized experiments, the amplification products of LAMP can be detected under 7 temperature gradients from 57°C to 63°C. The experimental results show that RT-LAMP-LFD does not require strictly controlled temperature. In the time-optimized experiment, the presence of the reaction product was clearly detected by agarose gel electrophoresis from 35 min, and saturation was observed after 50 min. Therefore, in order to save time in practical applications, the reaction time of LAMP can be controlled within a more flexible time range.

Primer design is the key to establishing RT-LAMP detection methods. Good primers can ensure the specificity of the detection method and the accuracy of the detection results. This experiment screened RT-LAMP primers designed based on the SVA 3D gene. The results indicate that the specific primers designed based on the SVA 3D gene have good specificity. Related studies have shown that the reaction conditions and systems of RT-LAMP, such as primer concentration, Mg2+ concentration and enzyme concentration, can directly affect the specificity and sensitivity of the detection[15]. Therefore, this experiment optimized the system and conditions of the established SVA RT-LAMP detection method, thus ensuring the high specificity and sensitivity of the detection method. In addition, the SVA RT-LAMP-LFD assay was found to have a higher clinical detection rate than RT-PCR in a clinical disease test.

In this study, a combination of RT-LAMP technology and LFD technology was applied to the detection of SVA and can now be used to analyze clinical samples such as SVA-infected tissues, vesicles and serum. The SVA RT-LAMP-LFD assay established in this trial aims to become a rapid and convenient diagnostic method for normal or suspected SVA-infected pigs at sites in the field. Its application value mainly lies in the initial diagnosis of SVA-infected pigs, the rapid screening of SVA in import and export trade, and the removal of SVA from pig farms. SVA RT-LAMP-LFD is a promising new tool for diagnosing SVA infection and is expected to have good application prospects.

Supporting information

S1 Fig. The alignment results of 3D regions base on the thirty-one full-length SVA sequences.

The figure shows the location of RT-LAMP primer binding sites within 3D genes.

https://doi.org/10.1371/journal.pone.0216245.s001

(TIF)

Acknowledgments

This experiment was jointly participated in by the National Institute of Veterinary Medicine of the South China Agricultural University, the National and Local Joint Engineering Laboratory for Zoonosis and Control Agents and the Key Laboratory of Veterinary Biotechnology Research and Application Enterprises of Guangdong Province. We thank the Guangdong Provincial Pig Industry System Innovation Team Fund for funding. All authors have seen the final version of this manuscript and approved it for publication. We ensure that this manuscript is not published in whole or in part elsewhere.

References

  1. 1. Zhao X, Wu Q, Bai Y, Chen G, Zhou L, Wu Z, et al. Phylogenetic and genome analysis of seven senecavirus A isolates in China. Transbound Emerg Dis. 2017;64(6):2075–82. %/ (c) 2017 Blackwell Verlag GmbH. pmid:28177200.
  2. 2. Adams MJ, Lefkowitz EJ, King AM, Harrach B, Harrison RL, Knowles NJ, et al. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016). Arch Virol. 2016;161(10):2921–49. pmid:27424026.
  3. 3. Hales LM, Knowles NJ, Reddy PS, Xu L, Hay C, Hallenbeck PL. Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus. J Gen Virol. 2008;89(Pt 5):1265–75. pmid:18420805.
  4. 4. Leme RA, Zotti E, Alcantara BK, Oliveira MV, Freitas LA, Alfieri AF, et al. Senecavirus A: An Emerging Vesicular Infection in Brazilian Pig Herds. Transbound Emerg Dis. 2015;62(6):603–11. %/ (c) 2015 Blackwell Verlag GmbH. pmid:26398783.
  5. 5. Zhang X, Xiao J, Ba L, Wang F, Gao D, Zhang J, et al. Identification and genomic characterization of the emerging Senecavirus A in southeast China, 2017. Transbound Emerg Dis. 2018;65(2):297–302. %/ (c) 2017 Blackwell Verlag GmbH. pmid:29219246.
  6. 6. Wang H, Li C, Zhao B, Yuan T, Yang D, Zhou G, et al. Complete genome sequence and phylogenetic analysis of Senecavirus A isolated in Northeast China in 2016. Arch Virol. 2017;162(10):3173–6. pmid:28687920.
  7. 7. Zhang Z, Zhang Y, Lin X, Chen Z, Wu S. Development of a novel reverse transcription droplet digital PCR assay for the sensitive detection of Senecavirus A. Transbound Emerg Dis. 2019;66(1):517–25. %/ (c) 2018 Blackwell Verlag GmbH. pmid:30375741.
  8. 8. Qian S, Fan W, Qian P, Chen H, Li X. Isolation and full-genome sequencing of Seneca Valley virus in piglets from China, 2016. Virol J. 2016;13(1):173. pmid:27756396.
  9. 9. Leme RA, Oliveira T, Alfieri AF, Headley SA, Alfieri AA. Pathological, Immunohistochemical and Molecular Findings Associated with Senecavirus A-Induced Lesions in Neonatal Piglets. J Comp Pathol. 2016;155(2–3):145–55. %/ Copyright (c) 2016 Elsevier Ltd. All rights reserved. pmid:27473601.
  10. 10. Pasma T, Davidson S, Shaw SL. Idiopathic vesicular disease in swine in Manitoba. Can Vet J. 2008;49(1):84–5. pmid:18320985.
  11. 11. Dall AA, Otonel R, Leme RA, Alfieri AA, Alfieri AF. A TaqMan-based qRT-PCR assay for Senecavirus A detection in tissue samples of neonatal piglets. Mol Cell Probes. 2017;33:28–31. %/ Copyright (c) 2017 Elsevier Ltd. All rights reserved. pmid:28267624.
  12. 12. Resende TP, Marthaler DG, Vannucci FA. A novel RNA-based in situ hybridization to detect Seneca Valley virus in neonatal piglets and sows affected with vesicular disease. PLoS One. 2017;12(4):e0173190. pmid:28394893.
  13. 13. Gimenez-Lirola LG, Rademacher C, Linhares D, Harmon K, Rotolo M, Sun Y, et al. Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality. J Clin Microbiol. 2016;54(8):2082–9. %/ Copyright (c) 2016, American Society for Microbiology. All Rights Reserved. pmid:27225408.
  14. 14. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. pmid:10871386.
  15. 15. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun. 2001;289(1):150–4. %/ Copyright 2001 Academic Press. pmid:11708792.
  16. 16. Nagamine K, Kuzuhara Y, Notomi T. Isolation of single-stranded DNA from loop-mediated isothermal amplification products. Biochem Biophys Res Commun. 2002;290(4):1195–8. %/ 2002 Elsevier Science (USA). pmid:11811989.
  17. 17. Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loop-mediated isothermal amplification reaction using a nondenatured template. Clin Chem. 2001;47(9):1742–3. pmid:11514425.
  18. 18. Osman HA, Eltom KH, Musa NO, Bilal NM, Elbashir MI, Aradaib IE. Development and evaluation of loop-mediated isothermal amplification assay for detection of Crimean Congo hemorrhagic fever virus in Sudan. J Virol Methods. 2013;190(1–2):4–10. %/ Copyright (c) 2013 Elsevier B.V. All rights reserved. pmid:23542058.
  19. 19. Wang C, Shen X, Lu J, Zhang L. Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for rapid detection of HDV genotype 1. Lett Appl Microbiol. 2013;56(3):229–35. %/ (c) 2012 The Society for Applied Microbiology. pmid:23252739.
  20. 20. Dukes JP, King DP, Alexandersen S. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus. Arch Virol. 2006;151(6):1093–106. pmid:16453084.
  21. 21. Whiting SH, Champoux JJ. Properties of strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase: mechanistic implications. J Mol Biol. 1998;278(3):559–77. %/ Copyright 1998 Academic Press Limited. pmid:9600839.
  22. 22. Howson E, Kurosaki Y, Yasuda J, Takahashi M, Goto H, Gray AR, et al. Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus. J Virol Methods. 2017;249:102–10. %/ Copyright (c) 2017 The Authors. Published by Elsevier B.V. All rights reserved. pmid:28837842.
  23. 23. Li C, Chen J, Shi H, Zhang X, Shi D, Han X, et al. Rapid detection of porcine kobuvirus in feces by reverse transcription loop-mediated isothermal amplification. Virol J. 2014;11:73. pmid:24755372.
  24. 24. Kim EM, Jeon HS, Kim JJ, Shin YK, Lee YJ, Yeo SG, et al. Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination. J Vet Sci. 2016;17(3):421–5. pmid:26726027.
  25. 25. Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol. 2003;41(6):2616–22. pmid:12791888.
  26. 26. Chow WH, McCloskey C, Tong Y, Hu L, You Q, Kelly CP, et al. Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile. J Mol Diagn. 2008;10(5):452–8. pmid:18669881.
  27. 27. Chowdry VK, Luo Y, Widen F, Qiu HJ, Shan H, Belak S, et al. Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field. J Virol Methods. 2014;197:14–8. %/ Copyright (c) 2013 Elsevier B.V. All rights reserved. pmid:24300833.
  28. 28. Waters RA, Fowler VL, Armson B, Nelson N, Gloster J, Paton DJ, et al. Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection. PLoS One. 2014;9(8):e105630. pmid:25165973.
  29. 29. Tsai SM, Liu HJ, Shien JH, Lee LH, Chang PC, Wang CY. Rapid and sensitive detection of infectious bursal disease virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. J Virol Methods. 2012;181(1):117–24. %/ Copyright A(c) 2011 Elsevier B.V. All rights reserved. pmid:22353472.
  30. 30. Ge Y, Wu B, Qi X, Zhao K, Guo X, Zhu Y, et al. Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device. PLoS One. 2013;8(8):e69941. pmid:23936359.
  31. 31. Tsai SM, Chan KW, Hsu WL, Chang TJ, Wong ML, Wang CY. Development of a loop-mediated isothermal amplification for rapid detection of orf virus. J Virol Methods. 2009;157(2):200–4. pmid:19186192.
  32. 32. Bearinger JP, Dugan LC, Baker BR, Hall SB, Ebert K, Mioulet V, et al. Development and initial results of a low cost, disposable, point-of-care testing device for pathogen detection. IEEE Trans Biomed Eng. 2011;58(3):805–8. pmid:21342806.
  33. 33. Arunrut N, Suebsing R, Withyachumnarnkul B, Kiatpathomchai W. Demonstration of a very inexpensive, turbidimetric, real-time, RT-LAMP detection platform using shrimp Laem-Singh virus (LSNV) as a model. PLoS One. 2014;9(9):e108047. pmid:25255231.
  34. 34. Fowler VL, Ransburgh RH, Poulsen EG, Wadsworth J, King DP, Mioulet V, et al. Development of a novel real-time RT-PCR assay to detect Seneca Valley virus-1 associated with emerging cases of vesicular disease in pigs. J Virol Methods. 2017;239:34–7. %/ Copyright A(c) 2016 Elsevier B.V. All rights reserved. pmid:27916668.
  35. 35. Armson B, Walsh C, Morant N, Fowler VL, Knowles NJ, Clark D. The development of two field-ready reverse transcription loop-mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1. Transbound Emerg Dis. 2019;66(1):497–504. %/ (c) 2018 Blackwell Verlag GmbH. pmid:30372584.
  36. 36. Njiru ZK. Rapid and sensitive detection of human African trypanosomiasis by loop-mediated isothermal amplification combined with a lateral-flow dipstick. Diagn Microbiol Infect Dis. 2011;69(2):205–9. %/ Copyright A(c) 2011 Elsevier Inc. All rights reserved. pmid:21251567.
  37. 37. Kusumawati A, Tampubolon ID, Hendarta NY, Salasia SI, Wanahari TA, Mappakaya BA, et al. Use of reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for an easy and rapid detection of Jembrana disease virus. Virusdisease. 2015;26(3):189–95. pmid:26396986.
  38. 38. James HE, Ebert K, McGonigle R, Reid SM, Boonham N, Tomlinson JA, et al. Detection of African swine fever virus by loop-mediated isothermal amplification. J Virol Methods. 2010;164(1–2):68–74. %/ Copyright (c) 2009 Elsevier B.V. All rights reserved. pmid:19963011.
  39. 39. Foord AJ, Middleton D, Heine HG. Hendra virus detection using Loop-Mediated Isothermal Amplification. J Virol Methods. 2012;181(1):93–6. %/ Crown Copyright A(c) 2012. Published by Elsevier B.V. All rights reserved. pmid:22327143.
  40. 40. Tomlinson JA, Dickinson MJ, Boonham N. Rapid detection of Phytophthora ramorum and P. kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device. Phytopathology. 2010;100(2):143–9. pmid:20055648.