114网址导航114网址导航PMCID: PMC4835727Reverse Transcription Cross-Priming Amplification–Nucleic Acid Test Strip for Rapid Detection of Porcine Epidemic Diarrhea Virus,1 ,3 ,4 ,4 ,3 ,3 ,2 ,2 and
a,1,22Veterinary Research Institute, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Huhhot, Inner Mongolia, 010031, People’s Republic of China3(Sino-USA) SiChuan Nabii Bio-Tech Co., Ltd., Chengdu, SiChuan, 610041, People’s Republic of China4Ustar Biotechnologies (Hangzhou), Ltd., Hangzhou, Zhejiang, 310012, People’s Republic of ChinaaEmail:
Porcine epidemic diarrhea virus (PEDV) is a highly transmissible coronavirus that causes a severe enteric disease particularly in neonatal piglets. In this study, a rapid method for detecting PEDV was developed based on cross-priming amplification and nucleic acid test strip(CPA-NATS). Five primers specific for the N gene sequence of PEDV were used for the cross-priming amplification. Detection of amplification products based on labeled probe primers was conducted with strip binding antibody of labeled markers. The CPA method was evaluated and compared with a PCR method. The reverse transcription CPA system was further optimized for detecting PEDV RNA in clinical specimens. Results showed that the method was highly specific for the detection of PEDV, and had the same sensitivity as PCR, with detection limit of 10−6 diluted plasmid containing the target gene of PEDV. It was also successfully applied to detecting PEDV in clinical specimens. The reverse transcription CPA-NATS detection system established in this study offers a specific, sensitive, rapid, and simple detection tool for screening PEDV, which can contribute to strategies in the effective control of PEDV in swine.Optimization of primer combinationsThe optimization of amplification involved primer combinations with a high sensitivity and no false positive. A set of primers suitable for CPA detection of PEDV were designated by CPR-2, DF5B1, DF5F-12, F3-1, and B3-1 (). DF5F-12 and DF5B1 were labeled with FAM and biotin, respectively. Primer combination, CPR-2, DF5B1, DF5F-12, F3-1, and B3-1 gave a high sensitivity with no false positive ( up). Gel electrophoresis results below the strip graph confirmed the CPA reaction corresponding to the results on the strip ( down).CPA-NATS and gel electrophoresis results of different components and conditions optimization: (a) external primers, (b) probe, (c) cross primer, (d) dNTP, (e) MgSO4, (f) betaine, (g) Bst DNA polymerase, (h) reaction time of 60min, (i) reaction ...Primers and probes used in CPA-NATS of PEDV.Optimization of CPA assayOptimization of CPA assay included concentration optimizations of external primers, probe and cross primer. Using concentrations ranging from 0.1L to 0.6L, the optimal concentrations of external primers were determined to be 0.1L of 20M F3-1 and 0.2L of 20M B3-1 ().Probes were optimized by testing concentrations (from 0.2L to 0.8L) of one probe against a fixed concentration of another probe. The optimal concentrations were 0.3L and 0.6L for 20M DF5F-12 and 20M DF5B1, respectively ().The optimal concentration of the cross primer was 0.4L of 20M CPR-2 after testing with concentrations ranging from 0.1L to 1L. (). Other reagents for the assay including MgSO4, betaine, deoxy-ribonucleoside triphosphate (dNTP), and Bst DNA polymerase were also optimized. The final volumes of dNTP (10mM), MgSO4 (50mM), betaine (5M), and Bst DNA polymerase (10U/L) were 0.8, 0.4, 6, and 1L, respectively (). A reaction temperature of 63°C was selected for the CPA assays, similar to previous report. The reaction time was extended by 15 and 30min on the basis of the optimum experimental conditions to determine the relationship between reaction time and sensitivity. Our results indicated that, when CPA reactions were incubated for 90min, favorable results were obtained without nonspecific amplification (). The sensitivity of CPA was higher than that under 60min () and 75min (). In addition, amplification results from the optimized CPA assay correlated 100% with those based on gel electrophoresis.Finally, the total volume for the CAP amplification was 20L. The optimal concentration of each reagent was as follows: CPR-2, 0.4M; F3-1, 0.1M; B3-1(20M), 0.2M; DF5F(20M), 0.3M; DF5B(20M), 0.6M; MgSO4, 2mM; betaine (5M), 1.5M; dNTP(10mM), 0.8L; and 1×Bst DNA polymerase buffer and Bst DNA polymerase (10U/L), 1L. The assay was able to detect plasmid containing PEDV target at 10−4 dilution after 60min amplification and at 10−6 dilution after 90min. No false positive was detected after 120min incubation.Reverse transcriptioncross-priming amplification (RT CPA)An assay combining RT and CPA was optimized to detect PEDV RNA from clinical specimens. AMV (BioFlux, China), Gsp Fast polymerase (Ustar, China), and RNA secure (Thermo Fisher, USA) in the RT CPA system were optimized. The optimal content of each component in the RT CPA system was as follows: CPR-2 (20M), 0.4M; F3-1 (20M), 0.1M; B3-1 (20M), 0.2M;DF5F (20M), 0.3M; DF5B(20M), 0.6M; MgSO4(50mM),2mM; betaine (5M), 1.5M; dNTP(10mM), 0.08M; Gsp Fast polymerase (10U/L), 0.2U/L; RNA secure, 1L; and AMV (5U/L), 0.05U/L, 1×Gsp Fast polymerase buffer, 2L. The total volume was 20L.Sensitivity and specificity of reverse transcription cross-priming amplification and nucleic acid test strip (RT CPA-NATS) systemTen times dilution series of plasmid was tested using CPA-NATS to determine its sensitivity. At the same time, reverse transcription PCR(RT-PCR) was used to confirm the CPA-NATS results. The results showed that 10−6 diluted plasmid could be detected consistently by CPA-NATS. However, results with 10−7 diluted plasmid were inconsistent (). Thus, 10−6 was considered the detection limit of CPA-NATS, which had a similar sensitivity compared to that of RT-PCR ().Sensitivity and specificity of RT CPA-NATS and RT-PCR: (a) sensitivity of CPA-NATS. N means negative control. −5, −6, −7 means 10−5, 10−6, 10−7 dilution. The detection limit was defined to 10−6 dilution, ...The specificity of the RT CPA-NATS method for detecting PEDV was evaluated by testing other common pathogenic viruses of swine, including porcine circovirus virus (PCV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine pseudorabies virus (PRV). Data showed 100% specificity against these viruses (), comparable to RT-PCR.Evaluation and validation of RT CPA-NATS with clinical specimensA total of 41 fecal samples from swine suspected of PEDV infections collected in Chengdu, Harbin, and Jinan were tested for further evaluation and validation of the RT CPA-NATS in the detection of PEDV. Ten samples (No. 5, 13, 21, 22, 23, 32, 35, 36, 37, and 38) were detected positive by RT CPA-NATS (). By contrast, only seven samples (No. 21, 22, 23, 29, 35, 37, and 38) were detected positive by RT-PCR (). The test results of the clinical specimens by RT CPA-NATS and RT-PCR were also summarized in . The number of PEDV-positive samples detected by RT CPA-NATS was 2 of 20 in Chengdu, 3 of 11 in Harbin, and 5 of 10 in Jinan (). However, two samples (one from Chengdu and one from Jinan) were detected positive by RT CPA-NATS but negative by RT-PCR. Another sample from Harbin was detected positive by RT-PCR but negative by RT CPA-NATS.Evaluation of 41 clinical specimens from Chengdu, Harbin, and Jinan by RT CPA-NATS: (a) RT CPA-NATS data and (b,c) gel electrophoresis data. The product of RT-PCR is approximately150bp. N, P, positive control. Samples 1–20 ...The test results of clinical specimens by RT CPA-NATS and RT-PCR.Data from application of the RT CPA-NATS on clinical specimens.Additionally, the system was validated by technicians with limited laboratory trainings on a pig farm. Six fecal swab specimens from sick piglets showing disease symptoms Suspected infected by PEDV were collected and tested for PEDV. Two of the six specimens (2# and 3#) were tested positive (), which was in agreement with the results of RT-PCR ().Trial of RT CPA-NATS detecting clinical specimens: (a) result of PEDV RT CPA-NATS and (b) result of PEDV RT-PCR.Ethics statementThe methods were carried out in accordance with Animal Epidemic Prevention Law of the People’s Republic of China. Animal experiments in this study were approved by the animal care and use committee of the Institute of Special Economic Animal and Plant Sciences and the animal welfare committee of the Jilin Province, China.As one-step diagnostic system, RT CPA-NATS was capable of detecting PEDV in clinical specimens and displayed 100% specificity against several related porcine viruses. The CPA-NATS system based on an isothermal DNA amplification technology and does not require any sophisticated equipment. It has already been applied to clinical diagnosis of human infectious diseases such as tuberculosis, where a double crossing CPA was used to amplify DNA of Mycobacterium tuberculosis. The present study employing the CPA-NATS technology was a single crossing CPA assay, which is slightly different from a double crossing CPA. We successfully adopted the technology and optimize all reaction conditions to meet our objective in developing diagnostic system that combines reverse transcription and CPA for detecting RNA virus.Several methods for the detection and diagnosis of PEDV have been described. Recently, a real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was reported to be easy to operate and simple to read results, and did not require sophisticated instruments,. However, RT-LAMP detects pathogens using reaction-turbidity as an indicator, which is sometimes difficult to determine without an instrument. The test strip result of CPA-NATS offers direct visualization by eyes, and is much easier to be interpreted. A variety of PCR methods have been developed to detect PEDV, such as a nanoparticle-assisted PCR assay, duplex real-time RT-PCR, and multiplex TaqMan probe-based real-time PCR, which can detect and differentiate the variant and virulent strains of PEDV currently reported to be circulating in the US. The above mentioned molecular biology methods all need expensive instruments, which limits their clinical applications particularly in field laboratories or on farm.The RT CPA-NATS system developed in the present study was capable of detecting PEDV in clinical specimens and distinguishing PEDV from other common viral pathogens causing pig diseases, namely, PCV, TGEV, CSFV, PRV, and PRRSV. It has a good specificity for detection of PEDV with the RT CPA-NATS methods. That is same as previous developed RT-PCR, real time RT-PCR or RT-LAMP,,. By the contrast of RT CPA-NATS and RT-PCR, we found that RT CPA-NATS had a little better effect on detecting PEDV than RT-PCR and meanwhile it had a convenient and cheaper test procedure. In the previous research, ELISA and RT-PCR, RT-PCR and real time RT-PCR, RT-LAMP and real time RT-PCR were all compared with in virus detection. Different methods detecting the different viruses may show variable effect. Here, we have showed the comparison of RT CPA-NATS with RT-PCR on detecting PEDV. However, the system was incapable of discriminating variants and virulent strains of PEDV. Nevertheless, it offers an excellent tool for initial screening of clinical specimens for PEDV, and should make valuable contributions to the rapid identification of PEDV in order to prevent and control this devastating disease in swine.In conclusion, our data showed that the RT CPA-NATS system developed for the detection of PEDV has significant advantages. The system was optimized and was comparable to RT-PCR that is the current recommended molecular method. Greater specificity and sensitivity, easy operation protocol and simple equipment make the RT CPA-NATS system ideal for the rapid detection of PEDV in clinical diagnosis.PrimersLarge fragment Bst DNA polymerase (Invitrogen, Beijing, China) was used in amplification reaction. According to the mechanism of CPA, the presence of a cross primer is essential. The two pairs of outer primers, three upstream probes, four downstream probes, and three cross primers were designed for screening the best primer combinations. The primers that target the N gene of PEDV were designed based on a new PEDV sequence (KC, NT 26716&#x). The optimum primers and probes () selected to develop CPA-NATS of PEDV were synthesized by Sangon Biotech (Shanghai, China). The probes were labeled with FAM or biotin.Plasmid as positive templateA constructed plasmid T-PEDV-N was used in developing CPA-NATS as positive template. Total RNA of PEDV was extracted with the E.A.N.A.™ Viral RNA Kit (OMIGA, Norcross, GA) as template to conduct RT-PCR to amplify the PEDV N gene fragment. The primers N-F/R were used to amplify PEDV N gene fragment (sequences were listed in ). The amplicon was cloned into pMD18-T vector(Sangon Biotech, Shanghai, China) by TA cloning at site 425 and called T-PEDV-N. The concentration of T-PEDV-N was 241.5g/mL, with approximately 5.39×1013 copies/mL.CPA-NATS procedureWe conducted a probe hybridization reaction experiment and determined the selected primers that exhibited no reaction during probe hybridization. Subsequently, the CPA tests were conducted with different primer combinations. The initial test system was as follows: CPR(20M), 0.8L; F3(20M), 0.2L; B3(20M), 0.2L; DF5F(20M), 0.3L; DF5B(20M), 0.3L; Thermo Pol buffer (10×), 2L; MgSO4(100mM), 0.4L; betaine (5M), 2L; dNTP(10mM), 0.8L; Bst DNA polymerase (10U/L), 1L; and template, 4L; sterile deionized water was added to achieved 20L. The products of CPA were diluted with1×saline sodium citrate buffer and dropped onto the sample pad of the test strips (Ustar Biotech, Hangzhou, China). The test strips were observed after 1min. Positive PEDV is denoted by two red lines, with the top line for quality control and the bottom line for test. Only the control line is negative. Otherwise, only the test line is invalid.Specifity of CPA-NATSWe further evaluated the specificity of the CPA method for the detection of PEDV by testing other viruses, such as PCV, TGEV, PRV, and PRRSV, which cause similar symptoms in pigs. The genome (RNA or DNA) was extracted with the E.A.N.A.™ Viral RNA Kit (OMIGA, Norcross, GA) or E.A.N.A.™ Viral DNA Kit (OMIGA, Norcross, GA). The viruses were detected by the reverse transcription CPA-NATS (RT CPA-NATS) system.Optimization of RT CPA-NATSIn order to detect the PEDV RNA from clinical specimens, a system including RT and CPA was optimized. AMV reverse transcriptase(BioFlux, Hangzhou, China) was used due to its tolerance to high temperature even at 65°C. Bst DNA polymerase was changed to Gsp Fast polymerase (Ustar Biotech, Hangzhou, China) to adapt to RNA detection. The optimization condition of all components, except for AMV, Gsp Fast polymerase, and RNA secure, was retained. AMV, Gsp Fast polymerase, and RNA secure in the RT CPA-NATS system were optimized.RT CPA-NATS used for detection of PEDV in clinical specimensWe collected 41 viscera samples from pigs apparently infected with PEDV from 3 regions (Chengdu, Harbin, and Jinan) of China to evaluate the PEDV RT CPA-NATS method. Samples 1–20 were obtained from Chengdu, 21–31 from Harbin, and 32–41 from Jinan. We tested the 41 specimens using the constructed PEDV RT CPA-NATS method. In the same manner, RT-PCR tests with outer primers pair of CPA were conducted on these specimens to confirm CPA.Nonprofessional personnel in a livestock farm conducted the RT CPA-NATS to detect PEDV in the samples to verify the clinical use effect of the RT CPA-NATS method. The same samples were tested using their developed RT-PCR method with the detection primers D-800-F/R, which match M and N gene of PEDV, respectively (). The product length was 800bp.How to cite this article: Wang, F.-X. et al. Reverse Transcription Cross-Priming Amplification–Nucleic Acid Test Strip for Rapid Detection of Porcine Epidemic Diarrhea Virus. Sci. Rep. 6, 24702; doi: 10.1038/srep2).This work was supported in part by the Special Fund for Agro-scientific Research in the Public Interest () and the Nabii Bio crosswise tasks (201401).Author Contributions F.-X.W., Y.-N.J. and Y.-J.W. conceived and designed the study and wrote the manuscript. D.-Y.Y. and Z.-Y.S. conducted the experiments and analyzed the data. Q.H., S.-H.Z., S.-B.Z. and L.H. contributed to the interpretation of the results and participated in the critical revision of the manuscript. All authors read and approved the final manuscript.Ojkic D.
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