Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies
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The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.
KeywordsFusarium Molecular methods Fungal infection Filamentous fungi
This work was supported by JST/JICA and SATREPS, Grant No. Unicamp 02P-29548-09. M.S. has received a master degree scholarship from the São Paulo Research Foundation (FAPESP), Grant No. FAPESP 2011/16205-5.
Limitations of the Study
The DNA microarray, LAMP and real-time PCR techniques utilized in this study only discriminated FSSC from Fusarium spp. However, the real value would be to differentiate FSSC from FOSC, rather than discriminate the components inside the complex. In this respect, the results are still valid and have significance in the decision making for therapeutic purposes.
Nucleotide Sequence Accession Numbers
The sequences determined in this study were deposited in NCBI database with the Accession Numbers: AB817198.1, AB817200.1, AB817204.1, AB817208.1, AB817209.1, AB817210.1, AB817216.1, AB817221.1, AB817222.1, AB817225.1, AB817226.1, AB817227.1, AB817231.1 and KM099396 to KM099400, KU974270, KY020039 and KY020040.
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