Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis
Backgound: Conventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2019-10-01
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| Series: | Journal of Microbiology, Immunology and Infection |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1684118218305012 |
| _version_ | 1818200164778639360 |
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| author | Ming-Horng Tsai Lee-Chung Lin Jen-Fu Hsu Mei-Yin Lai Hsuan-Rong Huang Ming-Chou Chiang Jang-Jih Lu |
| author_facet | Ming-Horng Tsai Lee-Chung Lin Jen-Fu Hsu Mei-Yin Lai Hsuan-Rong Huang Ming-Chou Chiang Jang-Jih Lu |
| author_sort | Ming-Horng Tsai |
| collection | DOAJ |
| description | Backgound: Conventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD). Methods: We evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification. Results: We have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively. Conclusions: Universal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease. |
| first_indexed | 2024-12-12T02:33:19Z |
| format | Article |
| id | doaj.art-c17f8c54fba14cc483dd6d60294e8ddb |
| institution | Directory Open Access Journal |
| issn | 1684-1182 |
| language | English |
| last_indexed | 2024-12-12T02:33:19Z |
| publishDate | 2019-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Microbiology, Immunology and Infection |
| spelling | doaj.art-c17f8c54fba14cc483dd6d60294e8ddb2022-12-22T00:41:21ZengElsevierJournal of Microbiology, Immunology and Infection1684-11822019-10-01525728735Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysisMing-Horng Tsai0Lee-Chung Lin1Jen-Fu Hsu2Mei-Yin Lai3Hsuan-Rong Huang4Ming-Chou Chiang5Jang-Jih Lu6Division of Neonatology and Pediatric Hematology/Oncology, Department of Pediatrics, Chang Gung Memorial Hospital, Yunlin, Taiwan; College of Medicine, Chang Gung University, Taiwan; Graduate Institute of Clinical Medical Science, College of Medicine, Chang Gung University, TaiwanDepartment of Laboratory Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan, TaiwanDivision of Pediatric Neonatology, Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan, Taiwan; College of Medicine, Chang Gung University, TaiwanDivision of Pediatric Neonatology, Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan, Taiwan; College of Medicine, Chang Gung University, TaiwanDivision of Pediatric Neonatology, Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan, Taiwan; College of Medicine, Chang Gung University, TaiwanDivision of Pediatric Neonatology, Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan, Taiwan; College of Medicine, Chang Gung University, TaiwanDepartment of Laboratory Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan; Corresponding author. Department of Laboratory Medicine, Chang Gung Memorial Hospital, 5, Fu-Shing St., Kwei-Shan, Taoyuan, 333, Taiwan. Fax: +886 3 3971827.Backgound: Conventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD). Methods: We evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification. Results: We have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively. Conclusions: Universal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease.http://www.sciencedirect.com/science/article/pii/S1684118218305012 |
| spellingShingle | Ming-Horng Tsai Lee-Chung Lin Jen-Fu Hsu Mei-Yin Lai Hsuan-Rong Huang Ming-Chou Chiang Jang-Jih Lu Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis Journal of Microbiology, Immunology and Infection |
| title | Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis |
| title_full | Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis |
| title_fullStr | Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis |
| title_full_unstemmed | Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis |
| title_short | Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis |
| title_sort | rapid identification of invasive fungal species using sensitive universal primers based pcr and restriction endonuclease digestions coupled with high resolution melting analysis |
| url | http://www.sciencedirect.com/science/article/pii/S1684118218305012 |
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