Development Of Loop-mediated Isothermal Amplification For Rapid Detection Of Haemophilus Parasuis

Haemophlius parasuis(Hps)is harmful,it is one of the most common diseases of pig industry.In order to successfully control it,we have to establish a rapid diagnostic method and take appropriate preventive measures in time.But it is difficult to isolate Haemophilus suis.The conventional detection methods are cumbersome and require expensive experimental instruments.These methods are difficult to promote at the grassroots level.Therefore,it is necessary to establish a method for rapid detection of Haemophilus influenzae by using loop mediated isothermal amplification(LAMP).Its potential for application in clinical testing will be large.This study compared the published Hps 16 S rRNA sequences in GengBank,and find out the highly conserved region.A set of four specific primers were designed based on six particular regions of the sequence: The two outer primers F3,B3 and two inner primers FIP,BIP.LAMP for Hps was performed by strand displacement using the Bst DNA polymerase.The method for rapid detecting Hps by LAMP was established by optimizing the reaction conditions and the reaction system.In addition,the sensitivity and specificity of the method were tested.1.Screening of the reaction systemThe single-factor gradient was set to optimize the concentration ratios of outer and inner primers(F3:FIP,B3:BIP),the final concentration of Mg2+ and the final concentration of dNTPs.The gradient of primer ratio is 1:1,1:2,1:4,1:6,1:8 and 1:10.The gradient of Mg2+ concentration is 0 m M,2 mM,4 mM,6 mM,8 m M and 10 mM.The gradient of dNTPs is 0 mM,1 mM,2 mM,3 mM,4 mM and 5 mM.The optimum reaction system was established.In this reaction system,the concentration ratios of outer and inner primers was 1:4,the final concentration of Mg2+ was 6 mM,the final concentration of d NTPs was 3 mM,2.5 ?L of 10 xThermoPol buffer,1 ?L of Bst DNA polymerase,1 ?L of template,supply water to 25 ?L.2.Optimization of reaction conditionsThe single-factor gradient was set to optimize reaction temperature and reaction time.The gradient of reaction temperature is 60 ?,61 ?,62 ?,63 ?,64 ?,65 ? and 66 ?.The gradient of reaction time is 30 min,40 min,50 min,60 min,and 70 min.The results showed that the reaction can be completed at 63 ? for just 1 hours.3.Sensitivity detection of LAMPThe DNA of Hps was ten-fold serially diluted.The sensitivities of LAMP and PCR for detecting Hps were compared by using DNA with different dilution as template respectively.The sensitivity test showed that the sensitivity of LAMP is 100 times greater than that of PCR.The LAMP could detect the DNA templates as low as 0.241 pg/?L.4.Specificity detection of LAMPThe specificity of LAMP was tested by using Hps,Streptococcus suis,Salmonella enteriditis,Escherichia coli,Bacillus subtilis,Actinobacillus pleuropneumoniae and staphylococcus aureus as templates respectively.The results showed that only with Hps as template can positively detect by LAMP,while no LAMP products were obtained when detecting other strains,which showed the good specificity of the method.In this study,A rapid method for the detection of Hps by LAMP was successfully established.The method is simple,sensitive and specific.It can observe the results of the experiment visually and applies to rapid field diagnosis.