Mitochondrial DNA screening by melting curve analysis using peptide nucleic acid probes.

Analysis of single nucleotide polymorphisms (SNPs) in mitochondrial (mt)DNA hypervariable regions (HV) 1/2 is valuable in forensic investigations. We developed a method for mtDNA screening of the HV1 and HV2 regions by melting curve analysis, using peptide nucleic acid (PNA) probes. This method focuses on melting peak patterns obtained by thermal dissociation of PNA/DNA duplexes in amplified mtDNA products. Five PNA probe sets were designed to detect 25 SNPs in the two HV regions. We also detected non-target SNPs based on unexpected melting temperature (Tm) shifts. In fact, 62 SNPs (42 SNPs in HV1 and 20 in HV2) were identified, including the 25 target SNPs. Using this method, 46 melting peak patterns, including 8 pattern groups, were obtained in 60 unrelated individuals. The peak patterns were compared to 55 haplotypes identified by Sanger sequencing. The results obtained from analysis of target mtDNA SNPs were entirely consistent with those obtained by Sanger sequencing. Screening the HV1 and HV2 regions of mtDNA by this method may help minimize unnecessary recourse to full sequence analysis, allows to rapidly exclude samples that do not match evidence and reference samples, and may reduce turnaround times and analysis costs. Overall, this method may be effective and helpful in forensic investigations.

Effect of X-irradiation on Citrus Canker Pathogen Xanthomonas citri subsp. citri of Satsuma Mandarin Fruits.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most important bacterial diseases of citrus. Because citrus canker is not found in many countries including European Union and Australia, Xcc is strictly regulated in order to prevent its spread. In this study, the effects of X-irradiation on Xcc growth either in the suspension or on the surface of citrus fruits were investigated. The suspension containing 1×10(7) cfu/ml of Xcc was irradiated with different absorbed doses of X-irradiation ranging from 50 to 400 Gy. The results showed that Xcc was fully dead at 400 Gy of X-irradiation. To determine the effect of X-irradiation on quarantine, the Xcc-inoculated citrus fruits were irradiated with different X-ray doses at which Xcc was completely inhibited by an irradiation dose of 250 Gy. The D10 value for Xcc on citrus fruits was found to be 97 Gy, indicating the possibility of direct application on citrus quarantine without any side sterilizer. Beside, presence of Xcc on the surface of asymptomatic citrus fruits obtained from citrus canker-infected orchards was noted. It indicated that the exporting citrus fruits need any treatment so that Xcc on the citrus fruits should be completely eliminated. Based on these results, ionizing radiation can be considered as an alternative method of eradicating Xcc for export of citrus fruits.

A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes.

ABO genotyping has been routinely used to identify suspects or unknown remains in crime investigations. Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection and is based on melting temperature shifts due to thermal denaturation. In the present study, we developed a new method for ABO genotyping using peptide nucleic acid (PNA) probe-based FMCA. This method allowed for the simultaneous detection of three single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 526, and 803) and the determination of 14 ABO genotypes (A/A, A/O01 or A/O02, A/O03, B/B, B/O01 or B/O02, B/O03, O01/O01 or O01/O02 or O02/O02, O01/O03 or O02/O03, O03/O03, A/B, cis-AB01/A, cis-AB01/B, cis-AB01/O01 or cis-AB01/O02, and cis-AB01/cis-AB01). Using this method, we analyzed 80 samples and successfully identified ABO genotypes (A/A [n=5], A/O01 or A/O02 [n=23], B/B [n=3], B/O01 or B/O02 [n=18], A/B [n=9], O01/O01 or O01/O02 or O02/O02 [n=20], cis-AB01/A [n=1], and cis-AB01/O01 or cis-AB01/O02 [n=1]). In addition, all steps in the method, including polymerase chain reaction, PNA probe hybridization, and FMCA, could be performed in one single closed tube in less than 3h. Since no processing or separation steps were required during analysis, this method was more convenient and rapid than traditional methods and reduced the risk of contamination. Thus, this method may be an effective and helpful tool in forensic investigations.

A putative APSES transcription factor is necessary for normal growth and development of Aspergillus nidulans.

The nsdD gene encoding a GATA type transcription factor positively controls sexual development in Aspergillus nidulans. According to microarray data, 20 genes that were upregulated by deleting nsdD during various life cycle stages were randomly selected and deleted for functional analysis. None of the mutants showed apparent changes in growth or development compared with those of the wild-type except the AN3154 gene that encodes a putative APSES transcription factor and is an ortholog of Saccharomyces cerevisiae swi4. Deleting AN3154 resulted in retarded growth and development, and the gene was named rgdA (retared growth and development). The rgdA deletion mutant developed a reduced number of conidia even under favorable conditions for asexual development. The retarded growth and development was partially suppressed by the veA1 mutation. The conidial heads of the mutant aborted, showing reduced and irregular shaped phialides. Fruiting body development was delayed compared with that in the wild-type. The mutant did not respond to various nutritional or environmental factors that affected the development patterns. The rgdA gene was expressed at low levels throughout the life cycle and was not significantly affected by several regulators of sexual and asexual development such as nsdD, veA, stuA, or brlA. However, the rgdA gene affected brlA and abaA expression, which function as key regulators of asexual sporulation, suggesting that rgdA functions upstream of those genes.

Simple identification of veA1 mutation in Aspergillus nidulans.

The veA gene plays an important role in development of a homothallic filamentous fungus Aspergillus nidulans. The veA1 phenotype can be difficult to distinguish from the wild-type veA. Despite the importance of the veA allele, no efficient identification method has been reported besides DNA sequencing. Here, we present simple physiological and molecular biological ways to distinguish between the veA wild-type and veA1 allele. The novel approaches, which involve incubation in the presence of oxalic acid, polymerase chain reaction using double mismatched primers, and BstXI enzyme digestion, are simpler, faster and more cost-efficient than genome sequencing.