Abstract: Yan Zhenzhen, Jiang Haiqin, Sun Jianfang, Shen Yongnian, Cui Pangen, Liu Weijun, Wang Hongsheng, Liu Weida. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: Wang Hongsheng, Email: whs33@vip.sina.com; Liu Weida, Email: liumyco@hotmail.com 【Abstract】 Objective To develop a microplate-reverse hybridization method combined with PCR-enzyme-linked immunosorbent assay （"simulant chip"） for the detection and identification of common species of mycobacteria and fungi in lesions of cutaneous infectious granuloma. Methods Two universal primers for mycobacteria and fungi, as well as specific probes for 10 common pathogens of cutaneous infectious granuloma （including Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, Mycobacterium fortuitum, Sporotrichum schencki, Cladosporium carrionii, Fonseceea pedrosoi, Fonseceea monophora and Candida albicans）, were designed. To test the performance of the universe primers, PCR was performed with them to amplify corresponding fragments from the standard strains of the 10 pathogens followed by sequencing. A "simulant chip" method was developed with the specific probes, and used to detect 10 standard and 19 clinical strains of these pathogens in simulated tissue specimens. The sensitivity and specificity of this method were determined. Results The corresponding fragments were amplified from all the standard strains by PCR with the two pairs of universal primers, and these PCR products were confirmed to match with the expected genes. As the "simulant chip" method showed, the absorbance value was 0.041 ± 0.02 for negative specimens, and the cut-off point was determined as 0.101 for this method. At this cut-off point, the detection limit of the "simulant chip" method was 1 × 101 to 1 × 102 cells/ml for these pathogens. No cross reaction was observed for these specific probes between these 10 pathogens. The genotyping results for these clinical isolates were consistent between the "simulant chip" method and DNA sequencing. Conclusion The established "simulant chip" method is a rapid method for the detection and identification of common pathogens from cutaneous infectious granuloma.

Key words: Granuloma, Mycobacterium infections, Fungal infections, Oligonucleotide reverse hybridization