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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

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, because 10−7 dilution plasmid was detected out randomly. (b) specificity of RT CPA-NATS. N: Negative control. The pathogens used to detect the specificity of RT-NATS and RT-PCR were CSFV, PRRSV, PCV, TGEV, PRV and PEDV. (c) sensitivity of RT-PCR. M: DNA Ladder DL2000. Lane 1 and 2 is negative control. Line 3–8 is 10−1~10−6. The detection limit was 10−6 dilution. The products size is about 150 bp. (d) gel electrophoresis confirmation of RT CPA-NATS. M: DNA Ladder DL2000. Lane 1: Negative control. Lane 2~Lane 8: PEDV, CSFV, PRRSV, PCV, TGEV, and PRV.
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f2: 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, because 10−7 dilution plasmid was detected out randomly. (b) specificity of RT CPA-NATS. N: Negative control. The pathogens used to detect the specificity of RT-NATS and RT-PCR were CSFV, PRRSV, PCV, TGEV, PRV and PEDV. (c) sensitivity of RT-PCR. M: DNA Ladder DL2000. Lane 1 and 2 is negative control. Line 3–8 is 10−1~10−6. The detection limit was 10−6 dilution. The products size is about 150 bp. (d) gel electrophoresis confirmation of RT CPA-NATS. M: DNA Ladder DL2000. Lane 1: Negative control. Lane 2~Lane 8: PEDV, CSFV, PRRSV, PCV, TGEV, and PRV.

Mentions: Ten 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 (Fig. 2a). Thus, 10−6 was considered the detection limit of CPA-NATS, which had a similar sensitivity compared to that of RT-PCR (Fig. 2c).


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)

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, because 10−7 dilution plasmid was detected out randomly. (b) specificity of RT CPA-NATS. N: Negative control. The pathogens used to detect the specificity of RT-NATS and RT-PCR were CSFV, PRRSV, PCV, TGEV, PRV and PEDV. (c) sensitivity of RT-PCR. M: DNA Ladder DL2000. Lane 1 and 2 is negative control. Line 3–8 is 10−1~10−6. The detection limit was 10−6 dilution. The products size is about 150 bp. (d) gel electrophoresis confirmation of RT CPA-NATS. M: DNA Ladder DL2000. Lane 1: Negative control. Lane 2~Lane 8: PEDV, CSFV, PRRSV, PCV, TGEV, and PRV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: 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, because 10−7 dilution plasmid was detected out randomly. (b) specificity of RT CPA-NATS. N: Negative control. The pathogens used to detect the specificity of RT-NATS and RT-PCR were CSFV, PRRSV, PCV, TGEV, PRV and PEDV. (c) sensitivity of RT-PCR. M: DNA Ladder DL2000. Lane 1 and 2 is negative control. Line 3–8 is 10−1~10−6. The detection limit was 10−6 dilution. The products size is about 150 bp. (d) gel electrophoresis confirmation of RT CPA-NATS. M: DNA Ladder DL2000. Lane 1: Negative control. Lane 2~Lane 8: PEDV, CSFV, PRRSV, PCV, TGEV, and PRV.
Mentions: Ten 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 (Fig. 2a). Thus, 10−6 was considered the detection limit of CPA-NATS, which had a similar sensitivity compared to that of RT-PCR (Fig. 2c).

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