| Bovine mastitis is a common disease which seriously restricts the healthy and orderly development of dairy farming.This disease has serious harm and complicated causes,among which pathogenic bacteria infection is the main cause of bovine mastitis.Generally,it takes a period of time from pathogen infection to the occurrence of bovine mastitis.If the infected pathogen can be detected early and accurately,and targeted early treatment and intervention can effectively reduce the incidence of mastitis It can be seen that early detection of pathogenic bacteria has positive significance for early diagnosis and early treatment of bovine mastitis,especially recessive mastitis.There are many types of pathogenic microorganisms that cause mastitis in dairy cows,among Streptococcus is one of the most important pathogenic bacteria that cause mastitis,mainly Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis are the most common.At present,the detection methods commonly used for these three kinds of streptococcus mainly include microbiological cultivation,serological detection and PCR methods,etc.However,the above methods generally have the characteristics of time-consuming,poor sensitivity,and high requirements for equipment and operation,which are not conducive to field promotion and application.However,the above methods generally have such disadvantages as time consuming,low sensitivity,and high requirements for equipment and operation,which are not conducive to field promotion and application.Loop mediated isothermal amplification has many advantages such as rapid,sensitive,specific,simple operation and low cost.It has become the most promising gene diagnosis technology and has been widely used in various research fields.In this study,the specific sequences of Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis were screened.By designing and screening primers,optimizing the reaction system and reaction conditions,the visual LAMP for rapid detection of Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis were established,respectively,and we detected clinical samples,which can provide a an effective technology for the clinical detection of these three pathogens.The main experimental results are as follows:1.Through literature review and NCBI database comparison analysis,it was determined that the cfb gene(GenBank ID:HQ148672.1)of Streptococcus agalactiae,the 16S rRNA gene(GenBank ID:EF121447.1)of Streptococcus dysgalactiae,and the 16S rRNA(GenBank ID:KX389960.1)of Streptococcus uberis as the target gene.According to the highly conserved sequence of the target gene,multiple sets of LAMP primer sets are designed respectively,and finally a set of specific LAMP primers are screened for the three kinds of bacteria,respectively.2.By optimizing LAMP reaction system and reaction conditions and combining with the calcein fluorescent dye,visual LAMP detection methods for Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis were established.The best reaction temperature of the three methods is 65℃,and the best reaction time is 50 min,40 min and 30 min,respectively.3.The established visual LAMP detection method for the Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis can only detect target strains,and has no cross-reactivity with other common non-target strains;the minimum detection line can reach 5.0 × 101 copies/μL,which is two orders of magnitude higher than the PCR method,and has high specificity and sensitivity.4.A total of 300 milk samples were collected from Ningxia.Visual LAMP method for Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis established in this study was used to detect clinical samples respectively,and the results were compared with PCR detection results.Among the 300 clinical samples,the positive rate of visual LAMP for Streptococcus agalactiae was 42.0%,and that of PCR was 31.0%;the positive rate of visual LAMP for Streptococcus dysgalactiae was 32.3%,and that of PCR The positive rate of the method was 24.3%;the positive rate of Streptococcus uberis visual LAMP was 22.3%,and the positive rate of PCR was 10.3%.It further shows that the established visual LAMP detection method for the three bacteria still has high sensitivity in the detection of clinical samples,and has a good application prospect.Collectively,it suggested that visual LAMP assay established in this study for Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis has advantages of strong specificity,high sensitivity,simple operation,time saving,low cost,easy detection,and with a low requirement in experimental conditions,which is more suitable for rapid detection on-site. |
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