Porcine circovirus type 2 (PCV-2) is a virus of the Circoviridae family that since the first reports in the 90s has had an increasing impact on the swine industry in pig-producing countries all over the world causing multiple syndromes. Many diagnostic protocols have been developed and used for PCV-2 detection. Most of them are highly accurate but they can be costly and time-consuming, limiting their application, especially in developing countries. To develop and validate a rapid and inexpensive protocol that can find application for research purposes and routine diagnosis, a direct Real-Time PCR (without DNA extraction) for the detection of PCV-2 was compared to a standard Real-Time PCR, requiring a preliminary DNA extraction step. Optimization experiments for the direct Real-Time PCR were performed evaluating procedures on samples before loading and amplification like preheating, pipetting and centrifuging. Since no significant improvement in efficiency and sensitivity was detected after integrating these extra steps in the protocol, the final validation and comparison with the standard Real-Time PCR (with DNA extraction) was performed using the less time-consuming and easier direct protocol. The parallel comparison was important for validating the accuracy of the protocol. Statistical analysis showed that the standard Real-Time PCR method provides higher PCR efficiency and repeatability and a lower variability in the viral titre estimation than the direct method. The overall higher efficiency of the standard Real-Time method could be explained by the process of extraction that helps to reduce potential inhibitors of amplification reaction. The here validated direct Real-Time PCR method represents a potential alternative to the standard one for developing countries, guaranteeing a more affordable and rapid detection method to control PCV-2. On the other hand, the standard Real-Time PCR method still ensures higher efficiency and repeatability, thus more reliable results which are crucial when higher accuracy is needed. The optimization of the direct method in order to enhance its performances is a prospect for future research.

Porcine circovirus type 2 (PCV-2) is a virus of the Circoviridae family that since the first reports in the 90s has had an increasing impact on the swine industry in pig-producing countries all over the world causing multiple syndromes. Many diagnostic protocols have been developed and used for PCV-2 detection. Most of them are highly accurate but they can be costly and time-consuming, limiting their application, especially in developing countries. To develop and validate a rapid and inexpensive protocol that can find application for research purposes and routine diagnosis, a direct Real-Time PCR (without DNA extraction) for the detection of PCV-2 was compared to a standard Real-Time PCR, requiring a preliminary DNA extraction step. Optimization experiments for the direct Real-Time PCR were performed evaluating procedures on samples before loading and amplification like preheating, pipetting and centrifuging. Since no significant improvement in efficiency and sensitivity was detected after integrating these extra steps in the protocol, the final validation and comparison with the standard Real-Time PCR (with DNA extraction) was performed using the less time-consuming and easier direct protocol. The parallel comparison was important for validating the accuracy of the protocol. Statistical analysis showed that the standard Real-Time PCR method provides higher PCR efficiency and repeatability and a lower variability in the viral titre estimation than the direct method. The overall higher efficiency of the standard Real-Time method could be explained by the process of extraction that helps to reduce potential inhibitors of amplification reaction. The here validated direct Real-Time PCR method represents a potential alternative to the standard one for developing countries, guaranteeing a more affordable and rapid detection method to control PCV-2. On the other hand, the standard Real-Time PCR method still ensures higher efficiency and repeatability, thus more reliable results which are crucial when higher accuracy is needed. The optimization of the direct method in order to enhance its performances is a prospect for future research.

Does DNA extraction really matter? Validation and comparison of a classic and direct real-time PCR protocol for the rapid and economic diagnosis of Porcine circovirus 2 (PCV2) infection.

MATI, ERINA
2021/2022

Abstract

Porcine circovirus type 2 (PCV-2) is a virus of the Circoviridae family that since the first reports in the 90s has had an increasing impact on the swine industry in pig-producing countries all over the world causing multiple syndromes. Many diagnostic protocols have been developed and used for PCV-2 detection. Most of them are highly accurate but they can be costly and time-consuming, limiting their application, especially in developing countries. To develop and validate a rapid and inexpensive protocol that can find application for research purposes and routine diagnosis, a direct Real-Time PCR (without DNA extraction) for the detection of PCV-2 was compared to a standard Real-Time PCR, requiring a preliminary DNA extraction step. Optimization experiments for the direct Real-Time PCR were performed evaluating procedures on samples before loading and amplification like preheating, pipetting and centrifuging. Since no significant improvement in efficiency and sensitivity was detected after integrating these extra steps in the protocol, the final validation and comparison with the standard Real-Time PCR (with DNA extraction) was performed using the less time-consuming and easier direct protocol. The parallel comparison was important for validating the accuracy of the protocol. Statistical analysis showed that the standard Real-Time PCR method provides higher PCR efficiency and repeatability and a lower variability in the viral titre estimation than the direct method. The overall higher efficiency of the standard Real-Time method could be explained by the process of extraction that helps to reduce potential inhibitors of amplification reaction. The here validated direct Real-Time PCR method represents a potential alternative to the standard one for developing countries, guaranteeing a more affordable and rapid detection method to control PCV-2. On the other hand, the standard Real-Time PCR method still ensures higher efficiency and repeatability, thus more reliable results which are crucial when higher accuracy is needed. The optimization of the direct method in order to enhance its performances is a prospect for future research.
2021
Does DNA extraction really matter? Validation and comparison of a classic and direct real-time PCR protocol for the rapid and economic diagnosis of Porcine circovirus 2 (PCV2) infection.
Porcine circovirus type 2 (PCV-2) is a virus of the Circoviridae family that since the first reports in the 90s has had an increasing impact on the swine industry in pig-producing countries all over the world causing multiple syndromes. Many diagnostic protocols have been developed and used for PCV-2 detection. Most of them are highly accurate but they can be costly and time-consuming, limiting their application, especially in developing countries. To develop and validate a rapid and inexpensive protocol that can find application for research purposes and routine diagnosis, a direct Real-Time PCR (without DNA extraction) for the detection of PCV-2 was compared to a standard Real-Time PCR, requiring a preliminary DNA extraction step. Optimization experiments for the direct Real-Time PCR were performed evaluating procedures on samples before loading and amplification like preheating, pipetting and centrifuging. Since no significant improvement in efficiency and sensitivity was detected after integrating these extra steps in the protocol, the final validation and comparison with the standard Real-Time PCR (with DNA extraction) was performed using the less time-consuming and easier direct protocol. The parallel comparison was important for validating the accuracy of the protocol. Statistical analysis showed that the standard Real-Time PCR method provides higher PCR efficiency and repeatability and a lower variability in the viral titre estimation than the direct method. The overall higher efficiency of the standard Real-Time method could be explained by the process of extraction that helps to reduce potential inhibitors of amplification reaction. The here validated direct Real-Time PCR method represents a potential alternative to the standard one for developing countries, guaranteeing a more affordable and rapid detection method to control PCV-2. On the other hand, the standard Real-Time PCR method still ensures higher efficiency and repeatability, thus more reliable results which are crucial when higher accuracy is needed. The optimization of the direct method in order to enhance its performances is a prospect for future research.
Real-Time PCR
Porcine circovirus 2
Extraction
Validation
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12608/34581