Development Of Quantitative Real-time PCR Assay For Diagnosis Of Penicilliosis Marneffei

Objective To develop a quantitative real-time PCR assay for early andspecific diagnosis of human penicilliosis marneffei in serums.Methods A pair of oligonucleotide primers and a TaqMan Probe, basedon a highly conserved ITS1-5.8S rDNA gene sequence of Penicillium marneffeiafter alignment of sequences using GeneBank and molecular biology software(Clustalx, Bio Edit), were designed by Primer express3.0software. PurifiedPCR products, obtained with above primers by conventional PCR, were clonedinto the pEASY-T5Zero vector. Sequencing was performed on recombinantplasmids in both directions to confirm the target sequences amplified. Therecombinant plasmids, quantified and converted into copy number, were used toestablish the standard curve which can quantify the initial fungal burden ofsamples in this study. A standard curve for qPCR system was generated usingtenfold serial dilution of purified DNA plasmid. This investigation also includedthe evaluation of sensitivity and specificity. Besides, DNA of clinical samples was detected and quantified by this assay.Results The successful design of specific primers and probe were used toamplification. The standard curve of real-time PCR was completed:Y=-3.34X+43.22,R2=0.994, and the slope was about-3.3. The real-time PCR Procedure wasable to deetect at least10copies per action, and had no cross-reaction withhuman genomic DNA, other fungus and baeterium.The first serum of55patientswith PSM were positive, and the Cqvalues are between28.39to39.65.Conclusion The quantitative real-time PCR assay was stable, reliable, andcontribute to early, specific diagnosis of PSM.