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Rapid and Simple Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) by Reverse Transcription Loop-Mediated Isothermal Amplification

Rapid and Simple Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) by Reverse Transcription Loop-Mediated Isothermal Amplification

Xi-wen Chen1,2, Qian Wang1,3, Miao Yin1, Zhong-hui Pu1,2, Ai-wei Guo3, Lian Li 1,3, Wen-tao Luo1,3 and Xiong-qing Wang1,*

1Institute of Applied Animal Technology, Mianyang Normal University, Mianyang 621000, China
2Research Center of Ecological Agriculture and Animal Husbandry in Northwest Sichuan, Mianyang, 621000, China
3College of Life Science, Southwest Forestry University, Kunming, 650224, China

*      Corresponding author: wangxq193@163.com

 

Fig. 1.

Verification of the PRRSV RT-LAMP assay. RT-LAMP was used to amplify a positive control template and compared to a negative control. The output of the RT-LAMP assay was monitored by gel electrophoresis and color change. (A) 1% agarose gel electrophoresis of the RT-LAMP products. Lanes: M; DL2000 Marker, 1; negative control, 2; positive control. (B) Color change in the RT-LAMP reaction was monitored followig addition of SYBR Green I dye. Tube 1: negative control, Tube 2: positive control. (C) The positive and negative controls were verified by RT-PCR 1% agarose gel electrophoresis of the RT-PCR products. Lanes: M; DL2000 Marker, 1; negative control, 2; positive control.

Fig. 2.

Optimizing the duration of the RT-LAMP reaction. RT-LAMP was used to amplify PRRSV containing samples. The products of the RT-LAMP reactions were visualized by (A) 1% agarose gel electrophoresis, (B) UV transillumination, and (C) color change using SYBR Green I dye. Lanes M; DL2000 Marker (A only), 1-5; 45, 50. 55, 60, and 65 min, respectively.

Fig. 3.

Optimizing the temperature of the RT-LAMP reaction. RT-LAMP was used to amplify PRRSV containing samples. The products of the RT-LAMP reactions were visualized by (A) 1% agarose gel electrophoresis. (B) UV transillumination, and (C) color change using SYBR Green I dye. Lanes: M; DL2000 Marker (A only), 1-5; 59, 61, 63, 65, and 67 C, respectively.

Fig. 4.

Assessing the specificity of RT-LAMP for PRRSV. Samples were isolated from pigs infected with PRRSV, CSFV, PCV-2, PPV, and PRV and tested usig the RT-LAMP assay. The output of the assay was visualized using (A) agarose gel electrophoresis and (C) color change with SYBR Green I dye. (B) The presence of PRRSV in the samples was confirmed by RT-PCR and visualized using agarose gel electrophoresis. Lanes: M; DL2000 Marker (A and B only). 1; CSFV, 2; PCV-2, 3; PPV, 4; PRRSV, 5; PRV.

Fig. 5.

Assessing the sensitivity of the PRRSV RT-LAMP assay. 10-fold serial dilutions of a stock PRRSV RNA template (10ng/μL) were used to assess the sensitivity of the RT-LAMP assay compared to RT-PCR. Output from the RT-LAMP assay was visualized by (A) agarose gel electrophoresis and (C) color change with SYBR Green I dye. (B) RT-PCR amplification of PRRSV RNA was visualized by agarose gel electrophoresis. Lanes: M; DL2000 Marker (A and B only); 1-5: 10-1, 10-2, 10-3, 10-4, and 10-5 dilutions of the stocktemplate.

Fig. 6.

Detection of PRRSV infection in serum samples. Five (5) serum samples were tested for PRRSV infection using RT-LAMP and RT-PCR. The output of the RT-LAMP assay was visualized using (A) agarose gel electrophoresis, (C) UV transillumination, and (D) color change with SYBR Green I dye. (B) RT-PCR products were visualized using agarose gel electrophoresis of RT-PCR products. Lanes M; DL2000 Marker (A and B only); 1-5; serum samples.

Pakistan Journal of Zoology

August

Vol. 50, Iss. 4, Pages 1199-1600

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