Establishment of a real time PCR method for detecting Sporothrix brasiliensis

Abstract

Abstract:

Objective To establish a rapid, specific and sensitive real-time PCR method for the detection of Sporothrix brasiliensis. Methods The internal transcribed spacer (ITS) sequence of S. brasiliensis were downloaded from the NCBI database. The conserved regions were obtained by software comparison. Specific primers and probes were designed and synthesized to establish and optimize the real time PCR method. The optimized method was used to evaluate amplification efficiency, sensitivity, and specificity using standard concentration nucleic acids. The method in this study was evaluated by mouse infection model of S. brasiliensis and compared with culture. Results The detection limit of the real-time PCR method established in this study for S. brasiliensis was 100 fg. The method was negative for the amplification of S. shenckii, S. globosa, 28 other common pathogenic fungi, 3 common bacteria, human genome and mouse genome, and the specificity was 100%. The detection results of the method and culture of brain, liver, lung, spleen, kidney and lymph nodes in mice infected with S. brasiliensis were consistent. Conclusion The real time PCR method established in this paper is a rapid, sensitive and specific method for the identification of S. brasiliensis. It can also detect specimens of infected mouse models, which is helpful for early diagnosis of sporotrichosis caused by S. brasiliensis.

Key words: real-time PCR, Sporothrix brasiliensis, identification, animal infectious model

CLC Number:

ZHANG Ming-rui, ZHOU Ying, LIFu-qiu, YAO Chun-li, YANG Xin, GONG Jie, ZHAO Fei. Establishment of a real time PCR method for detecting Sporothrix brasiliensis[J]. Chinese Journal of Mycology, 2019, 14(5): 297-302.

References

[1] 中华医学会皮肤性病学分会真菌学组. 孢子丝菌病诊疗指南[J]. 中华皮肤科杂志, 2016, 49(7):456-459.
[2] Ramos-E-Silva M, Vasconcelos C, Carneiro S, et al. Sporotrichosis[J]. Clin Dermatol, 2015, 25(2):181-187.
[3] Marimon R, Cano J, Gené J, et al. Sporothrix brasiliensis, S. globosa, and S. mexicana, three new Sporothrix species of clinical interest[J]. J Clin Microbiol, 2007, 45(10):3198-3206.
[4] de Beer ZW, Duong TA, Wingfield MJ. The divorce of Sporothrix and Ophiostoma:solution to a problematic relationship[J]. Stud Mycol, 2016,83:165-191.
[5] Ishida K, de Castro RA,Borba dos Santos LP. Amphotericin B, alone or followed by itraconazole therapy, is effective in the control of experimental disseminated sporotrichosis by Sporothrix brasiliensis[J]. Med Mycol,2015,53(1):34-41.
[6] Borbasantos LP, Rodrigues AM, Gagini TB, et al. Susceptibility of Sporothrix brasiliensis isolates to amphotericin B, azoles, and terbinafine[J]. Med Mycol, 2015, 53(2):178-188.
[7] Castro RA,Kubitschek-Barreira PH, Teixeira PA, et al. Differences in cell morphometry, cell wall topography and Gp70 expression correlate with the virulence of Sporothrix brasiliensis clinical isolates[J]. Plos One, 2013, 8(10):e75656.
[8] Fernandes GF, Santos POD, Rodrigues AM, et al. Characterization of virulence profile, protein secretion and immunogenicity of different Sporothrix schenckii sensu stricto isolates compared with S. globosa and S. brasiliensis species[J]. Virulence, 2013, 4(3):9.
[9] Gremião ID, Miranda LH, Reis EG. Zoonotic epidemic of sporotrichosis:cat to human transmission[J]. PLoS Pathog. 2017, 13(1):e1006077.
[10] Freitas DF, Lima MA, de Almeida-Paes R. Sporotrichosis in the central nervous system caused by Sporothrix brasiliensis[J]. Clin Infect Dis. 2015, 61(4):663-664.
[11] Rosane OC, Natasha U, Alexandre CG, et al. Pulmonary cavitation and skin lesions mimicking tuberculosis in a HIV negative patient caused by Sporothrix brasiliensis[J]. Med Mycol Case Rep, 2013, 2:65-71.
[12] Mario León Silva-Vergara, Camargo ZPD, Silva PF, et al. Case report:disseminated sporothrix brasiliensis infection with endocardial and ocular involvement in an HIV-infected Patient[J]. Am J Trop Med Hyg, 2012, 86(3):477-480.
[13] Orofinocosta R, Macedo PMD, Andréa RBE. Hyperendemia of sporotrichosis in the Brazilian Southeast:learning from clinics and therapeutics[J]. Curr Fungal Infect Rep, 2015, 9(4):220-228.
[14] De Meyer EM, De Beer ZW, Summerbell RC, et al. Taxonomy and phylogeny of new wood-and soil-inhabiting Sporothrix species in the Ophiostoma stenoceras-Sporothrix schenckii complex[J]. Mycologia, 2008, 100(4):647-661.
[15] Madrid H, Cano J,Josepa Gené, et al. Sporothrix globosa, a pathogenic fungus with widespread geographical distribution[J]. Rev Iberoam Micol, 2009, 26(3):218-222.
[16] Manoel O, Rodrigo AP, Mauro M, et al. Phenotypic and molecular identification of Sporothrix isolates from an epidemic area of sporotrichosis in Brazil[J]. Mycopathologia, 2011, 172(4):257-267.
[17] Romeo O,Scordino F, Criseo G. New insight into molecular phylogeny and epidemiology of Sporothrix schenckii species complex based on calmodulin-encoding gene analysis of italian isolates[J]. Mycopathologia, 2011, 172(3):179-186.
[18] Zhang Z, Liu X, Lv X, et al. Variation in genotype and higher virulence of a strain of Sporothrix schenckii causing disseminated cutaneous sporotrichosis[J]. Mycopathologia, 2011, 172(6):439.
[19] Sindy H, Wen-Hung C,Shuen-Iu H, et al. Detection of Sporothrix schenckii in clinical samples by a nested PCR assay[J]. J Clin Microbiol, 2003, 41(4):1414-1418.
[20] Xu TH, Lin JP, Gao XH, et al. Identification of Sporothix schenckii of various mtDNA types by nested PCR assay[J]. Med Mycol, 2010, 48(1):161-165.
[21] Rodrigues,Messias A, Hoog D, et al. Genotyping species of the Sporothrix schenckii complex by PCR-RFLP of calmodulin[J]. Diagn Microbiol Infect Dis, 2014, 78(4):383-387.
[22] Rodrigues AM,Najafzadeh MJ, Hoog GSD, et al. Rapid Identification of emerging human-pathogenic Sporothrix species with rolling circle amplification[J]. Front Microbiol, 2015, 6:1385.
[23] Rodrigues AM,Hoog GSD, Camargo ZPD. Molecular diagnosis of pathogenic Sporothrix species[J]. PLoS Negl Trop Dis, 2015, 9(12):e0004190.
[24] 刘小荣, 张笠, 王勇平. 实时荧光定量PCR技术的理论研究及其医学应用[J]. 中国组织工程研究, 2010, 14(2):329-332.
[25] Lopes-Bezerra LM, Mora-Montes HM, Zhang Y, et al. Sporotrichosis between 1898 and 2017:The evolution of knowledge on a changeable disease and on emerging etiological agents[J]. Med Mycol, 2018, 56(suppl 1):126-143.