背景:孢子丝菌病是皮肤和皮下组织的慢性肉芽肿性感染,可通过淋巴扩散影响任何器官。孢子丝菌病感染的患病率正在增加,其治疗具有挑战性,因为没有统一和标准的诊断技术或抗真菌药物。控制进一步传播需要快速诊断。临床症状评估,组织学分析,血清学检测,和病原体培养都是孢子丝菌病诊断所必需的。然而,这些程序无法识别物种。安全的发展,可靠,和特定物种的诊断技术是必不可少的。
目的:建立一种新的实时荧光定量PCR快速诊断孢子丝菌病的方法并进行鉴定。
方法:在定量实时PCR分析中使用钙调蛋白(CAL)基因序列和内部转录间隔区(ITS)的多态性来鉴定S.globosa,S.申克,和非目标物种。
结果:实时定量PCR检测具有100%的灵敏度和特异性。检测限为6fg/μl。证实34份临床标本感染球藻,阳性检出率为100%。
结论:本研究开发的定量PCR技术是一种快速,准确,以及基于CAL序列和ITS多态性的球藻的靶向鉴定方法。它可用于及时的临床诊断,以鉴定孢子丝菌病患者临床标本中的球形链球菌。
BACKGROUND: Sporotrichosis is a chronic granulomatous infection of the skin and subcutaneous tissue that can affect any organ through lymphatic spread. The prevalence of sporotrichosis infections is increasing and its treatment is challenging as there are no unified and standard diagnostic techniques or antifungal medications. Controlling further spread requires a rapid diagnosis. Assessment of clinical symptoms, histological analysis, serological testing, and pathogen culture are all necessary for the diagnosis of sporotrichosis. However, these procedures are unable to identify the species. The development of safe, reliable, and species-specific diagnostic techniques is essential.
OBJECTIVE: To establish and evaluate a new quantitative real-time PCR assay for the rapid diagnosis of sporotrichosis and to identify relevant species.
METHODS: Polymorphisms in calmodulin (CAL) gene sequences and the internal transcribed spacer (ITS) were used in a quantitative real-time PCR assay to identify S. globosa, S. schenckii, and non-target species.
RESULTS: The quantitative real-time PCR assay had 100% sensitivity and specificity. The limit of detection was 6 fg/µl. Thirty-four clinical specimens were verified to be infected with S. globosa with a 100% positive detection rate.
CONCLUSIONS: The quantitative PCR technique developed in this study is a quick, accurate, and targeted method of identifying S. globosa based on polymorphisms in CAL sequences and ITS. It can be used for a prompt clinical diagnosis to identify S. globosa in clinical specimens from patients with sporotrichosis.