Abstract: Objective To optimize nested-PCR of Bacillus piliformis detection and to explore its application. Method The liver from rats infected by Bacillus piliformis RJ was used as the positive control. This sample was positive for Bacillus piliformis detected by indirect immunofluorescence(IFA) according to the GB/T 14926.10-2008《laboratory animal- Method for examination of Tyzzer's organism GB/T 14926.10-2008》. Nested-PCR of Bacillus piliformis established previously by our lab was optimized, including DNA extraction and PCR conditions. This optimized nested-PCR was applied to detect the Bacillus piliformis of 22 immunosuppressive rats. Results Phenol/chloroform DNA extraction method of liver was optimal; A specific 196 bp DNA fragment was obtained, the sensitivity of this method reached to 1 pg; Blast results showed that all the sequences of the specific DNA fragment shared 100% homology with the 16 S rRNA of Bacillus piliformis presented in Genbank (D14638.1).Twenty-two immunosuppressive rats were detected for Bacillus piliformis. The positive rate by IFA is 36.4%(8/22); The positive rate by nested-PCR is 59.1% (13/22indicating nested-PCR is more sensitive than IFA. Meanwhile all the positive samples detected by EFAwere also positive detected by nested PCR.Conclusion This optimized nested-PCR method has high sensitivity and specificity and can be applied to Bacillus piliformis detection.

Key words: Bacillus piliformis;Nested-PCR, IFA