Rapid Detection Of Streptococcus Pyogenes By Loop-mediated Isothermal Amplification

Streptococcus pyogenes is a common food-borne pathogens. S. pyogenes causes various infections, including scarlet fever, impetigo and acute pharyngitis in children. S. pyogenes can spread through food, water and air. Rapid and accurate identification is critical to control the infection. Common methods which used to detect S. pyogenes are culture isolation method, immunological methods and PCR. But there are some limitations of these methods. So we need a new method which more rapid, sensitive and convenient. Here, we want to develop a method(loop-mediated isothermal amplification, LAMP) for the specific detection of S. pyogenes. The assay used specifically primers to target within the spy1258 gene which encodes the putative transcriptional regulator of S. pyogenes. We choose the optimum primers and reaction temperature by studying. Then the specificity and sensitivity of the primers were determined. We detected 30 samples collected from Peking by LAMP we developed here. Then Positive samples were isolated by emm typing. The main conclusions:We designed 3 primers SP-36, SP-84 and SP-106 by Primer Explorer V4 software. DNA of S. pyogenes was used as template. The results of LAMP showed that the SP-106 primers are optimum primers. Target DNA was amplified well by LAMP at an isothermal temperature of 64 degrees centigrade. The results of specificity of LAMP showed that the primers had good specificity for Spy1258. The sensitivity of LAMP, with a detection limit of 1.486 pg/μl DNA, was 10-fold greater than that of PCR.30 strains of S. pyogenes were correctly identified by LAMP developed here. And these 30 strains of S. pyogenes were isolated four genotype by emm genotype. They are emm1、emm4、emm12 and emm58. The S. pyogenes of emm1 and emm12 have the largest proportion, they are 83.3%.Consequently, the LAMP described here is able to detect S. pyogenes and it is more sensitive and convenient, so it can save some time and resources. LAMP has the potential to become a useful tool for the rapid detection of S. pyogenes.