| Porcine circovirus type 2(PCV2)is a single-stranded negative stranded DNA virus with a diameter of about 17 nm and is the smallest animal virus at this stage.Post Weaning Multisystemic Wasting Syndrome(PMWS)caused by PCV2 virus has a rapid onset and widespread spread.It is difficult to cure the disease and has a high mortality rate,causing great harm to the pig breeding industry in China and even the world.PCV2 is often mixed with some viruses,such as porcine reproductive and respiratory syndrome virus,porcine parvovirus,toxoplasmosis,eperythrozoon and so on,which can promote the onset of related diseases.Typical symptoms of PMWS include progressive wasting,dyspnea,anorexia,lethargy,shaggy hair,and jaundice in 20%of pigs.Macroscopically,mesentery and inguinal lymphadenopathy and gray brown inflammation are most common.Other organs,such as liver,spleen,pancreas,stomach,small intestine and colon,are also swollen and necrotic.Due to the severe damage of PCV2 to the immune system of the pigs,it results in low immunity.And once there are other viruses,bacteria,protozoan infection in pigs,it will outbreak.In present,the disease has been widely prevalent in the United States,France,China,South Korea and other countries,causing a huge loss to the world's pig industry.At present,the diagnosis of PCV2 mainly relies on immunological detection technology and molecular biological detection technology.However,these detection methods generally have some limitations,such as low sensitivity,high cost,complex operation process and other factors,which limit the application of detection technology.Therefore,it is necessary to develop a fast,simple,sensitive and accurate method to detect PCV2 virus.Our laboratory has developed the porcine circovirus virus type 2(PCV2)LAMP on-site visualization rapid detection kit.In this experiment,we intend to further study the raw materials,the technology and reaction system and the analytical performance of the kit,so as to optimize the reaction conditions in all aspects of the kit,improving the detection performance of the kit.In addition,the preliminary declaration of drug certificates for PCV2 LAMP on-site visual qualitative detection kit was carried out.This research also make preparations for declaration of drug certificates and the application of other on-site visual detection technologies.The main contents and results are as follows:1.Study on the main raw materials of PCV2 LAMP field visual rapid detection kitThe purpose of this study was to study the main raw materials of PCV2 LAMP field visual rapid detection kit.Based on the PCV2 CP sequence published by GenBank,PrimerExplorer V5 was used to design LAMP primers in its conserved sequence region and optimizing the reaction conditions.A LAMP kit reagent blow-drying process was established to reduce the contamination of PCV2 virus and the operation steps of DNA amplification in field.And kits for the Bst enzyme and LAMP reaction buffer comparison study,Bst enzyme and LAMP reaction buffer quality identification study,kit positive reference product research and kit negative reference product research were carried out,respectively.The method was applied at a constant temperature of 61? for 40 min,and the PCV2 virus DNA was amplified with high efficiency and specificity.In this test,Bst enzymes and LAMP reaction buffers from A,B and C Molecular Biotechnology Co.,Ltd were selected for comparison research and quality identification research.The results showed that reagents from B Biotechnology Co.,Ltd were better,and the raw materials the manufacturer could maximize the detection performance of the kit.The gradient dilution of the positive reference for the kit was studied,and the results revealed that the reference had highly sensitive.In the study on negative reference--porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV)and swine fever virus(CSFV)of the kit,it had good specificity.This study could also make preparations for the declaration of drug certificates for PCV2 LAMP on-site visual qualitative detection kit and also provide an accurate and reliable tool for the diagnosis of PCV2.2.Study on the process and reaction system of PCV2 LAMP field visual rapid detection kitThe purpose of this study was to study the process and reaction system of the on-site visualized rapid detection kit for PCV2 LAMP.Based on the PCV2 CP sequence published by GenBank,PrimerExplorer V5 was used to design LAMP primers in its conserved sequence region and optimizing the reaction conditions.A LAMP kit reagent blow-drying process for the specific amplification of PCV2 virus DNA was established,and LAMP detection kits were developed.The sample storage time study,sample freeze-thaw study,sample transportation storage time study,reagent dosage study,sensitivity study and specificity study were performed for the kit,respectively.The method was applied at a constant temperature of 61? for 40 min,and the PCV2 virus DNA was amplified with high efficiency and specificity.In this experiment,the clinically positive samples of PCV2 virus were placed at 2-8?,-20? and-70?,respectively and experimental tests were conducted at intervals to determine the validity of the samples.PCV2 positive samples were used in the test to design the freezing-thawing times of 1,3,5,7 and 9 times respectively.The results suggested that the freezing-thawing times of the samples should not exceed 5 times.Using PCV2 positive samples and simulating the transport conditions at room temperature,the results suggest that the sample transport time does not exceed 4 days.The specificity and sensitivity of the kit were evaluated,and the results showed that the specificity of the test was good and the sensitivity was high,and it could reach 2 copies/uL.At the same time,the external environment and the system dosage were also studied.The results showed that the detection method established in this study was less affected by the external environment.This work laid a foundation for the work of drug identification application of PCV2 LAMP field visual rapid detection kit,and provided an accurate and reliable tool for the diagnosis of PCV2.3.Study on the analysis performance of PCV2 LAMP field visual rapid detection kitThe purpose of this study was to study the analytical performance of the on-site visualized rapid detection kit for PCV2 LAMP.Based on the PCV2 CP sequence published by GenBank,PrimerExplorer V5 was used to design LAMP primers in its conserved sequence region and optimizing the reaction conditions.A LAMP kit reagent blow-drying process for the specific amplification of PCV2 virus DNA was established.The LAMP detection kits were developed,and the sensitivity,specificity,accuracy and interference substances of the kit were studied,respectively.The method was applied at a constant temperature of 61? for 40 min,and the PCV2 virus DNA was amplified with high efficiency and specificity.In this study,PCV2 plasmid was diluted by gradient to obtain 104,103,102,101,5,2,1 copies/uL,and the results showed that the detection technology can reach 2 copies/uL.Porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV)and porcine fever virus(CSFV)were used in the experiment to product the specificity research,and the results showed that the specificity of this experiment was good.The accuracy of the test was evaluated with known clinical samples,and the experimental results show that the detection technology has high accuracy.PCV2 virus culture solution was mixed with blood and serum samples,and the results showed that blood and serum did not interfere with the detection of PCV2 positive samples.In the experiment,antibiotics such as chlortetracycline,oxytetracycline and amoxicillin were added into the reaction reagent,and the results showed that the selected antibiotics did not interfere with the detection of the kit.This work laid a foundation for the work of drug identification application of PCV2 LAMP field visual rapid detection kit,and provided an accurate and reliable tool for the diagnosis of PCV2. |
PDF Full Text Request