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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

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 60 min, (i) reaction time of 75 min, (j) reaction time of 90 min, and (k) agarose gel electrophoresis confirmations of the CPA reaction corresponding to the results on the strips. N, negative control.
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f1: 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 60 min, (i) reaction time of 75 min, (j) reaction time of 90 min, and (k) agarose gel electrophoresis confirmations of the CPA reaction corresponding to the results on the strips. N, negative control.

Mentions: The 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 (Table 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 (Fig. 1a up). Gel electrophoresis results below the strip graph confirmed the CPA reaction corresponding to the results on the strip (Fig. 1a down).


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)

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 60 min, (i) reaction time of 75 min, (j) reaction time of 90 min, and (k) agarose gel electrophoresis confirmations of the CPA reaction corresponding to the results on the strips. N, negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: 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 60 min, (i) reaction time of 75 min, (j) reaction time of 90 min, and (k) agarose gel electrophoresis confirmations of the CPA reaction corresponding to the results on the strips. N, negative control.
Mentions: The 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 (Table 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 (Fig. 1a up). Gel electrophoresis results below the strip graph confirmed the CPA reaction corresponding to the results on the strip (Fig. 1a down).

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