High-resolution Melting Approach To Detection And Identification Of Four Clinically Important Invasive Aspergillus Species

Invasive fungal infection is a life-threatening infections in immuno-compromised patients. In recent years, invasive fungal infection increased in oncology, human immunodeficiency-irus-infected and transplant patients,carrying a high mortality. Candida species is the most commonly pathogens clinically. However, there has also been a rise in the incidence of disease caused by Cryptococcus especially Cryptococcus neoformans, filamentous fungi especially Aspergillus species and many other opportunistic fungi. Immunity of patients and timely administration of effective antifungal agents largely influenced the outcomes of treatment. At present, amphotericin B, ketoconazole, fluconazole, posaconazole, caspofungin are the most common antifungal agents. As is reported, there is an increase in the number of aspergillus isolates with resistance to antifungal agents, particularly the azoles and amphotericin B. Consequently, it is necessary to develop a new assay for detection and identification of clinically important Aspergillus species which may contributes to the antifungal therapy.The basic principle of high-resolution melting curve analysis is real-time monitoring the combination of double-strand DNA fluorescent saturating binding dyes with the PCR product in heating process reflecting changes in fluorescence signal),so as to achieve the sequence analysis. There is no difference in the binding mechanism between the saturating DNA binding dyes and traditional fluorescent dyes. However, traditional fluorescent dyes such as Sybr Green are unsaturated fluorescent dyes which inhibiting PCR process partly. As a result, it is of low concentration in PCR mix, far below the number of dye binding sites of DNA double-strand structure. The DNA double-strand denatures with the increase in temperature, the fluorescent dye molecules are released, performing the change in fluorescence signal. When released, unsaturated fluorescent dyes will exhibit redistribution and recombine with other double-strand DNA structure in the blank sites, that create fluorescence signal distortion, which does not accurately reflect the denaturation process of double-strand DNA in the PCR mix. In conrast, the saturated fluorescent dye exhibit minimal redistribution during melting and do not inhibit PCR allowing a true reflection of the double-strand DNA structure denaturation and provide a future for sequence analysis.Unlabeled probes are included in the high-resolution melting curve analysis is an new application. Unlabelled probes are an oligonucleotide of20-40bp. The3’end of the probe must be blocked to prevent extension of the probe itself. Comparing with the traditional fluorescent probe, there is no fluorophores modified on unlabelled probe. By performing denaturation and renaturation programmes after the completion of PCR, the probe binds to the single-stranded product, forming a partial double-stranded structure, and denatures in the following heating process, which lead to changes in the fluorescence signal to generate a probe melting peak which is more sensitive and specific.In our study, we utilized high-resolution, unlabelled probe and asymmetric PCR to detect and identify the four clinically important Aspergillus species to develop a rapid, accurate, and economic assay.Part1High-resolution melting curve analysis of panfungal ITS2amplicons for detection and identification of the four clinically important Aspergillus species. MethodFind the sequences of18s rRNA,5.8s rRNA,28s rRNA of Cryptococcus, Candida, Aspergillus and Penicillium in GeneBank. Align their sequences with Clustal X software to get the conserved regions of fungal rRNA genes with no sequence homology with human genome. Targeting the internal transcribed spacer region in fungal ribosomal RNA gene, design and blast panfungal primers. Amplify ITS2region of the four aspergillus species with panfungal primers designed and perform high-resolution melting curve analysis on PCR product.Aspergillus fumigatus isolate ATCC96918, Aspergillus flavus isolate ATCC28539, Aspergillus niger isolate ATCC16404, Aspergillus terreus isolate ATCC1012, human genome DNA, and a variety of clinically common bacterial organisms were used to determine the specificity of the panfungal primers.Optimize the method in annealing temperature, primers concentration, and Mg2+concentration which affects PCR and high-resolution melting curve analysis largely.Construct vector cloning with the ITS2PCR product of Aspergillus fumigatus ATCC96918to estimate detection limits. The plasmid was diluted9concentration gradient with the ratio of1:10. Amplify and perform high-resolution melting curve analysis to determine the detection limit.Results1. Panfungal primers sequences are as follows:PrimerF-AGCGAAATGCGATAASTARTGTG, PrimerR-TCCTCCGCTTATTGATATGC, with the product length of335bp.2. The panfungal primers can amplify the four aspergillus species with no amplification of other organisms.3. The detection limit of panfungal PCR-HRM analysis can be10copies/μl.4. There is much difference between the four Aspergillus species in Tm and melting behavior.ConclusionsIt can be demonstrated that in this part, the four Aspergillus species can be identified by their melting curves without sequencing.If this method was applied to other clinically important fungi species, a database of standard high-resolution melting curves could be created. Just with PCR primers, unknown fungi strain DNA template, saturated dye LC Green and instrument which could perform high-resolution melting curve analysis, by comparing the melting curve of unknown fungal strains with the standard curves in the database, the unknown strains can be detected and identified rapidly, saving time, labor and money.Part2High-resolution melting curve analysis with unlabelled probes for detection and identification of the four clinically important Aspergillus species. MethodFour species-specific probes of the four aspergillus species were designed without intraspecific variation in ITS2regions of aspergillus fumigatus, aspergillus flavus, aspergillus niger, and aspergillus terreus in GeneBank and modified using amino-modified C6as blocking agent to prevent DNA polymerase extension. Evaluation of primers and probes was performed with Primer Express software, Oligo6.0software and Blastn online.Perform asymmetric PCR with panfungal primers designed in part1. Add specie-specific unlabelled probes after asymmetic PCR. Then perform denaturation and renaturation, unlabelled probes binds with sing-stranded DNA to form partial double-stranded structure which performing melting analysis then.Optimize the assay in annealing temperature, primers concentration, primers ratio, Mg2+concentration and probes concentration which may affects PCR and high-resolution melting curve analysis largely. Primers concentration was increased from0.1μmol/L to0.5μmol/L, the proportion of forward primer and reverse primer changed from1;2to1:20,2:1to20:1, probe concentration increased from0.05μmol/L to0.5μmol/L and Mg2+concentration, increased from0.5mmol/L to5mmol/LResults1、The final composition and condition are as follows:5×PrimeSTAR Buffer,4μl; Mg2+,1mmol/L, dNTP Mixture (2.5mmol/L),1.6μl; primer-F,0.25μmol/L primer-R,0.05μmol/L; PrimeSTAR HS DNA Polymerase,0.2μl; LC Green saturated dye,1μl; oligonucleotide probes of the four aspergillus species,0.25μmol/L respecitvely, DNA template,1ul and Sterile distilled water to20μl。Amplification conditions:98℃,10s;60℃,10s;72℃,18s,60×cycles。melting stage:initial melting temperature,55℃, final melting temperature,98℃, holding temperature,53℃。2、High-resolution melting curve analysis of the four Aspergillus species all have two melting peaks. It can be known from the melting beheavior of the amplicons of four kinds of Aspergillus species in part1that Melting peak between90℃and95℃is from the double-stranded product of the first dozens of cycles genetated by asymmetic PCR.Software evaluation shows that the probe Tm values higher than75℃. Therefore, the melting peak between82℃87℃is from the melting of the double-stranded structure of unlabeled probe and single-stranded DNA amplified by the reverse primer with relatively high concentration in the last dozens of cycles of asymmetric PCR in which the reverse primer of relatively low concentration is exhausted melting. There is obvious difference in the Tm values between the probe-peak and the doulble-stranded product-peaks.ConclusionsSince the ribosomal RNA gene internal transcribed spacer2contains both the variable region and moderately conserved sequence. According to the ITS2sequences of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger published in the GeneBank, it is found that there are not only the sequence differenced with the species but also within the species, ranging from1bp to5bp in the internal transcribed spacer2variable region. The nucleotide differences as low as1bp can be detected by high-resolution curve analysis with saturated fluorescent dye. Due to the presence of intraspecies sequence differences,the melting patterns and Tm of PCR products may complicate the interpretation of melting analysis for identification.We designed the four Aspergillus species specific probes in the ITS2variable region and modified the3’end of the probe using amino-modified C6to block extention. Asymmetic amplification was performed with different concentration of forward and reverse primers. As asymmetic PCR is completed, there are two kinds of PCR product, one is a normal double-stranded product produced in the first dozens of cycles of the PCR process, the other is a large number of single-stranded DNA produced by primer of relatively high concentration in the last tens of cycles of the PCR process after completely comsuming the primer of relatively low concentrationsAfter a denaturation-renaturation process, the unlabelled probes combined with the single-strand product produced in the last dozens of cycles forming a partial double-strand structure which will melt in the following heating process generating a probe-melting peaks. Identification of the four Aspergillus species could be made by analysis on the melting peaks of probe and product. Analysis of the the melting peaks of the four Aspergillus species probes facilitate the identification of Aspergillus species without melting curve database. More importantly, the introduction of the specie-specific unlabelled probes was able to overcome the deformation and offset caused by intraspecies difference in the internal transcribed spacer2variable region of Aspergillus, greatly improving the usefulness and accuracy. The3’end of the probe was blocked without any fluorophore can be a big cost savings.