RESUMO
A double-molecular beacons (DMB) based assay was developed for porcine circovirus 2(PCV2) detection. Two single-stranded DNA molecular beacons which could specifically hybridize with PCV2 genome DNA respectively in different sequence were designed according to the characteristics of the PCV2 genome sequences. The fluorescence signal was amplified 80 times by DMB, which was 2-4 times higher than that of single molecular beacon. Under the optimal conditions of 10 mmol/L MgCl2 , 20 mmol/L Tris-HCl (pH=8. 0), 40 ℃ and 30 min incubation time of DNA with DMB, the enlargement factor was increased linearly with DNA concentration over the range from 2 nmol/L to 200 nmol/L, with a detection limit of 1 nmol/L. The method was applied to detect PCV2 in genome of 18 swine fever samples and 8 PCV2 positive cases were found, which were confirmed by PCR method.
RESUMO
Objectives A new method for the detection of PCR ampli fied DNA in capillary electrophoresis and laser induced fluorescence (CE LIF) system based on molecular beacons hybridization is described.Methods The method is based on the hybridization of molecular beacons into the amplification product, then separating the hybridized product with CE LIF system. This method was applied to the detection of p73exon3, p53exon5 and p16exon2 for gastric carcinoma. Results Detected with CE LIF molecular beacons method, the wild type percent of p73exon3 is 100%, same as SSCP; p53exon5 is 60 0%, while SSCP is 63 3%, P=0 99; p16exon2 is 20 0%, same as SSCP(20 0%).Conclusion The results demonstrate that the specific peaks appeared in wild type samples while no peaks or unspecific peaks in mutation ones. The results showed correlation with the results of PCR SSCP. The method is able to develope as a clinical screening method for tumor suppressor genes to diagnose carcinoma.