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Published on: February 4, 2018
Identification of Penicillium marneffei in Paraffin-Embedded Tissue Using Nested PCR
Hanxiang Zeng1, Xiqing Li, Xiejie Chen
1Department of Dermatology, The Second Affiliated Hospital, Sun Yat-Sen University, 107 West Yanjiang Rd, Guangzhou 510120, China.
Abstract:
Penicillium marneffei is one of the unique thermally dimorphic fungi in Penicillium species that causes a disseminated, progressive and life threatening infection in immunocompromised patients. The diagnosis of Penicilliosis marneffei depends on culture that may delay the treatment due to the time-consuming process. In the present study, we evaluated the specificity and sensitivity of nested PCR to identify Penicillium marneffei from paraffin-embedded tissue. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA of Penicillium marneffei. The outer primers (RRF1 and RRH1) were specific to fungi. The inner primers (Pm1 and Pm2) were specific to Penicillium marneffei. The specific fragment of approximately 400 bp was amplified from all paraffin-embedded tissues from 14 patients with Penicilliosis marneffei and 10 bamboo rats. The detectable DNA concentration of single PCR and nested PCR were 14 pg/microl and 14 fg/microl, respectively. Further studies are required in order to use nested PCR for early diagnosis of the disease.
Insights
Nested PCR offers a sensitive method for detecting Penicillium marneffei, the fungus causing Penicilliosis marneffei. This molecular technique shows promise for faster diagnosis compared to traditional culture methods, aiding immunocompromised patients.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Penicillium marneffei causes life-threatening disseminated infections in immunocompromised individuals.
- Current diagnosis relies on culture, which is time-consuming and can delay treatment.
Purpose of the Study:
- To evaluate the specificity and sensitivity of nested PCR for identifying Penicillium marneffei in paraffin-embedded tissues.
Main Methods:
- Developed two sets of oligonucleotide primers targeting the 18S rRNA gene of Penicillium marneffei.
- Utilized outer primers specific to fungi and inner primers specific to Penicillium marneffei.
- Amplified a 400 bp fragment from infected tissues and bamboo rat samples.
Main Results:
- Nested PCR successfully amplified the target fragment from all 14 Penicilliosis marneffei patient tissues and 10 bamboo rats.
- Demonstrated high sensitivity with detectable DNA concentrations of 14 fg/microl for nested PCR versus 14 pg/microl for single PCR.
Conclusions:
- Nested PCR is a specific and sensitive method for detecting Penicillium marneffei in paraffin-embedded tissues.
- Further research is needed to establish nested PCR for early diagnosis of Penicilliosis marneffei.
