Isolation And Identification Of Bacillus Cereus And Establishment Of RPA-LF Method For Detection Of NHE Gene

A strain of bacteria was isolated from the spleen of a sudden dead beef cattle in our laboratory,which was identified as pathogenic Bacillus cereus.Since there are no reports about the death of cattle infected by Bacillus cereus at present,the strains were isolated and identified,and the RPA-LF detection method for the nhe gene of the bacteria was established,in order to provide a theoretical basis for the research,prevention and control of the disease.The bacteria were identified by morphological observation,biochemical detection,16s rDNA and groEL gene sequence analysis.Its biological characteristics were detected by drug sensitivity,acid tolerance,bile salt tolerance,high temperature tolerance,animal pathogenicity and pathological section observation of infected mice.After that,the virulence genes of the bacteria were detected and analyzed by PCR detection and base sequence determination.Then a rapid RPA-LF detection method was established for the virulence gene nhe of the strain.The results showed that the strain was Gram-positive bacilli with single or chain arrangement,biochemical test,16s rDNA and groEL gene analysis showed that the strain was Bacillus cereus,and the results of biological characteristics showed that the strain was sensitive to Cefalexin,gentamicin and norfloxacin and had acid tolerance and bile salt tolerance,but the high temperature tolerance was not strong compared with other Bacillus cereus.The results of virulence gene detection showed that the strain contained virulence genes hbl,nhe,entFM and cytk;.The best reaction temperature for RPA-LF detection of nhe gene was 38?,the lowest effective reaction time was 10 min,and the lowest effective detection concentration was 1.0 ×102 CFU·mL-1,which did not cross-react with Clostridium perfringens and other bacteria.The results of this study show that:The isolated bacteria are pathogenic Bacillus cereus and contain toxins hbl,nhe,cytk and entFM.A RPA-LF method for detection of nhe gene was successfully established,which is specific and sensitive.