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Rapid identification of Penicillium marneffei by PCR-based detection of specific sequences on the rRNA gene
Nongnuch Vanittanakom1, Pramote Vanittanakom, Roderick J Hay
1Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. nvanitta@mail.med.cmu.ac.th
Abstract:
An emerging pathogenic dimorphic fungus, Penicillium marneffei, is one of the major causes of morbidity in patients with human immunodeficiency virus infection in Southeast Asia. A PCR-hybridization assay has been developed to identify this pathogen. This study describes the use of single and nested PCR methods for the rapid identification of P. marneffei. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA genes of P. marneffei. The outer primers (RRF1 and RRH1) were fungus specific. The inner primers (Pm1 and Pm2) were specific for P. marneffei and were used in nested or single PCR. The specific fragment of approximately 400-bp was amplified from both mold and yeast forms of 13 P. marneffei human isolates, 12 bamboo rat isolates, and 1 soil isolate, but not from other fungi, bacteria, and human DNA. The amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The sensitivities of the single PCR and nested PCR were 1.0 pg/microl and 1.8 fg/microl, respectively. The assay is useful for rapid identification of P. marneffei cultures. Very young culture of P. marneffei (2-day-old filamentous colony, 2 mm in diameter) could be performed by this assay. The species was identified within 7 h (single PCR) or 10 h (nested PCR), compared to 4 to 7 days for confirmation of dimorphism. The application of these PCR methods for early diagnosis of the disease needs to be studied further.
Insights
A new PCR method rapidly identifies Penicillium marneffei, a fungus causing illness in HIV patients in Southeast Asia. This molecular assay offers faster diagnosis than traditional methods.
Area of Science:
- Medical Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Penicillium marneffei is a dimorphic fungus causing significant morbidity in HIV-infected individuals in Southeast Asia.
- Accurate and rapid identification of P. marneffei is crucial for timely clinical intervention.
- Existing diagnostic methods can be time-consuming, delaying treatment.
Purpose of the Study:
- To develop and evaluate a Polymerase Chain Reaction (PCR)-based assay for the rapid and specific identification of Penicillium marneffei.
- To assess the sensitivity and speed of single and nested PCR methods for P. marneffei detection.
Main Methods:
- Oligonucleotide primers were designed based on the 18S rRNA gene sequence of P. marneffei.
- Single and nested PCR assays were performed using specific primers (Pm1, Pm2) and fungus-specific outer primers (RRF1, RRH1).
- Amplified DNA fragments were analyzed by agarose gel electrophoresis and ethidium bromide staining.
Main Results:
- A specific 400-bp DNA fragment was successfully amplified from both mold and yeast forms of P. marneffei isolates from human, bamboo rat, and soil sources.
- The assay demonstrated high specificity, with no amplification observed from other fungi, bacteria, or human DNA.
- Nested PCR achieved a sensitivity of 1.8 fg/microl, significantly higher than single PCR (1.0 pg/microl).
- Identification was achieved within 7-10 hours, drastically reducing the time compared to traditional 4-7 day methods.
Conclusions:
- Single and nested PCR assays provide a rapid, sensitive, and specific method for identifying Penicillium marneffei.
- This molecular technique allows for the early detection of P. marneffei, even from very young cultures.
- Further studies are warranted to explore the application of these PCR methods for early disease diagnosis.
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