Application Of PCR And ELISA In The Detection Of Leptospira

Leptospirosis is a widespread,natural zoonotic disease caused by the pathogenic Leptospira.Animals such as pigs,horses,cattle,sheep,dogs and cats,as well as humans,can be infected with Leptospira,the symptoms include jaundice,miscarriage,liver and kidney failure,and even death in severe cases.At the same time,the infected animals continue to release the bacteria into surroundings,causing a wider spread of infection.Therefore,it is necessary to diagnose the disease as early as possible and implement effective coping strategies.Because the current PCR detection of Leptospira cannot detect all the existing pathogenic Leptospira in China,and because the MAT experiment has a relatively high operating threshold,not all laboratories can carry out it,which is not conducive to the early diagnosis of Leptospira.Based on these reasons,a PCR detection method for the pathogen of Leptospira and an ELISA detection method for its antibodies were established.So far,PCR detection of Leptospira is a commonly and mature used laboratory detection method.Studies have shown that pathogenic Leptospira contain a conserved fragment of the Lip L32 gene sequence.Based on this,three pairs of primers with the same annealing temperature were designed by comparing the Lip L32 gene sequences of the reference standard strain of 15serovars of 15 serogroups Leptospira in China.During this period,its specificity,sensitivity and repeatability have been repeatedly verified to ensure its efficient and accurate detection of Leptospira.In this experiment,the sensitivity of PCR to detect leptospira concentration was10^-6ng/?L.Then,on the basis of three pairs of primers,degenerate base primers were designed for fluorescence quantitative PCR detection,which was also verified by specificity,repeatability and sensitivity,so that it could be used for effective detection of actual samples.In the test,the CT value of 30 is the limit:when the CT value is less than 30,it is determined to be positive,otherwise,it is negative.Based on this,a PCR method,detection of Leptospira was established.Although the MAT(microscopic agglutination test)is the gold standard for the diagnosis of Leptospira,the identification of live Leptospira bacteria and related professionals is required.In view of some shortcomings of the microscopic agglutination test,in order to accurately determine the serogroups of Leptospira,this experiment established an ELISA detection method that can detect the serogroups of Leptospira.Firstly,live strains of 15 serogroups of 15 serovars Leptospira were immunized to rabbits according to the set immunization program.After the program,corresponding antiserums were obtained as detection antibodies.Serums to be tested were coated and matched with 15 kinds of inactivated Leptospira.The absorbance value was measured at the wavelength of 450nm,and it was determined to be positive when the OD value of the tested sample was twice that of the negative serum.The results showed that 9 out of 11 water samples were positive,13 out of 24 aborted cows were positive,and 1 out of 3 healthy cows showed a recessive state of carrying bacteria.For Nanchang sick dog Leptospira isolates of appraisal,using polymerase chain reaction(PCR)to identify the cultivated bacteria was Leptospira,meanwhile has carried on the MAT and ELISA to sickness dog serum,the results have shown the antibodies in the serum was most intense agglutination and the titer was highest,the results of the two methods are consistent.Based on this,through MLST analysis,it was finally concluded that it was Leptospira interrogans Serovar Australis.Intraperitoneal injection of this bacterium could cause the death of golden hamster,and it was found that the liver,kidney and lung had obvious pathological damage.The total lethal amount of Leptospira was 10⁸.PCR detection for Leptospira pathogen and ELISA detection for its antibodies can be effective for the differential diagnosis of Leptospira.This is not only conducive to the clinical diagnosis of Leptospira and the early detection of Leptospira,but also plays a certain role in the prevention and control of the disease.