ABSTRACT
Objective To develop a sensitive, specific, rapid and easy method for detecting E. coli O157 : H7 and other Shiga toxin-producing E. coli in food and clinical specimens. Methods A circular probe and capture probe specific for Shiga toxin-2 (stx2) gene had been synthesized and was used for determining the sensitivity of ramification amplification method (RAM). Different serotypes which contained stx2 gene, including an E. coli O157 : H7, an E. coli O46 : H38, an E. coli O111 : NM, an E. coli O22 : H8 and E. coli ATCC23716 (stx2 gene negative) were used for determining the specificity. All strains were detected by RAM to determine whether they contained stx2 gene. Real-time RAM was further studied to detect stx2 gene. Results The lowest number targets detected by RAM assay was 10 copies of stx2, indicating that RAM assay was as sensitive as conventional PCR. The result of specificity showed that different serotypes of strains were all positive for stx2 gene, while nonpathogenic E. coli ATCC23716 was negative. Among 32 isolates, 28 STEC isolates containing stx2 gene were positive by RAM assay, while others were negative. The RAM results were 100% in concordance with that of PCR. The real-time RAM results also showed that as many as 10 bacteria can be detected and the time of appearance of detectable signal was depended on the target concentration. Conclusion RAM assay can offer an alternative method for PCR to detect E. coli O157 : H7 and STEC in all types of specimens because of its simplicity and isothermal amplification.
ABSTRACT
Objective To detect EB virus in peripheral blood of patients with non-hodgkin lymphoma (NHL) by ramification amplification method (RAM).To investigate the relativity of NHL with infection of EB virus.Methods RAM was used to detect EB virus with the man-made target gene sequence of promotor of EBER-1. In 120 cases of patients with NHL, EB virus were detected by RAM, and the result of RAM was compared with that of PCR.Raji cells and NB4 cells were used as positive control and negative control respectively.Results RAM could detect 10 molecular target gene of the exponent.76 cases were positive in 120 cases of NHL patients and the positive rate was 63.3%, The result is identical with that of PCR.Conclusion Ramification amplification method is sensitive, convenient and easy to perform.NHL has a high relativity with infection caused by EB virus.