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Reverse Transcription Cross-Priming Amplification-Nucleic Acid Test Strip for Rapid Detection of Porcine Epidemic Diarrhea Virus.

Wang FX, Yuan DY, Jin YN, Hu L, Sun ZY, He Q, Zhao SH, Zhan SB, Wen YJ - Sci Rep (2016)

Bottom Line: The reverse transcription CPA system was further optimized for detecting PEDV RNA in clinical specimens.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Special Economic Animal Molecular Biology, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, No. 4899 Juye Avenue, Jingyue Economic and Technological Development Zone, Changchun, Jilin, 130112, People's Republic of China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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 approximately150 bp. N, negative control; P, positive control. Samples 1–20 were obtained from Chengdu, 21–31 from Harbin, and 32–41 from Jinan. Seven samples (Nos 21, 22, 23, 29, 35, 37, and 38) were tested PEDV positive by RT-PCR. However, 10 samples (Nos 5, 13, 21, 22, 23, 32, 35, 36, 37, and 38) were tested PEDV positive by RT CPA-NATS.
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f3: 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 approximately150 bp. N, negative control; P, positive control. Samples 1–20 were obtained from Chengdu, 21–31 from Harbin, and 32–41 from Jinan. Seven samples (Nos 21, 22, 23, 29, 35, 37, and 38) were tested PEDV positive by RT-PCR. However, 10 samples (Nos 5, 13, 21, 22, 23, 32, 35, 36, 37, and 38) were tested PEDV positive by RT CPA-NATS.

Mentions: A 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 (Fig. 3a). By contrast, only seven samples (No. 21, 22, 23, 29, 35, 37, and 38) were detected positive by RT-PCR (Fig. 3b–d). The test results of the clinical specimens by RT CPA-NATS and RT-PCR were also summarized in Table 2. 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 (Table 3). 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.


Reverse Transcription Cross-Priming Amplification-Nucleic Acid Test Strip for Rapid Detection of Porcine Epidemic Diarrhea Virus.

Wang FX, Yuan DY, Jin YN, Hu L, Sun ZY, He Q, Zhao SH, Zhan SB, Wen YJ - Sci Rep (2016)

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 approximately150 bp. N, negative control; P, positive control. Samples 1–20 were obtained from Chengdu, 21–31 from Harbin, and 32–41 from Jinan. Seven samples (Nos 21, 22, 23, 29, 35, 37, and 38) were tested PEDV positive by RT-PCR. However, 10 samples (Nos 5, 13, 21, 22, 23, 32, 35, 36, 37, and 38) were tested PEDV positive by RT CPA-NATS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835727&req=5

f3: 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 approximately150 bp. N, negative control; P, positive control. Samples 1–20 were obtained from Chengdu, 21–31 from Harbin, and 32–41 from Jinan. Seven samples (Nos 21, 22, 23, 29, 35, 37, and 38) were tested PEDV positive by RT-PCR. However, 10 samples (Nos 5, 13, 21, 22, 23, 32, 35, 36, 37, and 38) were tested PEDV positive by RT CPA-NATS.
Mentions: A 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 (Fig. 3a). By contrast, only seven samples (No. 21, 22, 23, 29, 35, 37, and 38) were detected positive by RT-PCR (Fig. 3b–d). The test results of the clinical specimens by RT CPA-NATS and RT-PCR were also summarized in Table 2. 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 (Table 3). 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.

Bottom Line: The reverse transcription CPA system was further optimized for detecting PEDV RNA in clinical specimens.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Special Economic Animal Molecular Biology, Institute of Special Economic Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, No. 4899 Juye Avenue, Jingyue Economic and Technological Development Zone, Changchun, Jilin, 130112, People's Republic of China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus