Development And Application Of LAMP For Theileria Sergenti

Theileriosis is one of blood protozoal disease, caused by parasitize erythrocyte and lymphocyte Theileria family, Theileria genus, Theileria sergenti, the symptom is fever,anaemia,bleeding,emaciation lymphaden is intumescentia. If the cows are ill, there are many symptoms, so that cause a lot of economical loss for cattle farming.A part of P33 gene sequences as target sequence, designing LAMP primers by Primer Explorer3.0 software. Optimized the parameters of temperature,Magnesium ion concentration,in and out primers concentration ratio,reaction time and so on. The result showed that when out primers concentration ratio keep invariant, Magnesium ion concentration is 8mM andlOmM in primers concentration is 1.2u M-1.6μM, we can get the best effect, so we can confirm temperature is 63℃,and out primers concentration is 0.2μM, obtained 2 bands after digesting by ScaⅠ(114bp and 129bp), the best time is 45min or 60min, T. S P33-LAMP minimum test value is 0.23fg/μl, but common PCR is only 23fg/u 1, the template of specificity test is Toxoplasma gondii,Neospora caninum,Ovate Babesia, there is no specificity lands;in sensitive experiment. Application test of 84 blood samples of 4 areas showed that they are infect areas, the highest positive rate is 85.71%, different sex is notable(P<0.05),and different areas is very notable (P<0.01). All the results indicated that the LAMP is a quick,sensitive,specific,good stability method, is good for prophase diagnose, is the base for T. SP33-LAMP kit.