Application and Prospect of RNA Profiling Analysis in Forensic Body Fluid Identification.

In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.

PCR-RFLP for Detection of Fusarium graminearum Genotypes with Resistance to Phenamacril.

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and F. asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in in vitro lab experiments. In this study, PCR restriction fragment length polymorphism (RFLP) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217; and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L), or 1,649 bp (for mutation of E420K) in the myosin-5 gene were amplified by appropriate primer pairs. Restriction enzyme KpnI, TasI, or DraI was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L, and E420K, respectively. KpnI digested the 841-bp PCR products of phenamacril-resistant strains with codon mutation A135T into two fragments of 256 and 585 bp. In contrast, KpnI did not digest the PCR products of sensitive strains. TasI digested the 802-bp PCR products of phenamacril-resistant strains with codon mutation S217L into three fragments of 461, 287, and 54 bp. In contrast, TasI digestion of the 802-bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515 and 287 bp. DraI digested the 1,649-bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three mutation genotypes of F. graminearum resistant to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

Resistance mechanism of Fusarium fujikuroi to phenamacril in the field.

BACKGROUND: Rice bakanae disease, mainly caused by Fusarium fujikuroi, is an important disease of rice. Phenamacril has been used to control the disease for a few years in China. In 2016, nine phenamacril-resistant strains were found in the field in Zhejiang Province. The aim of the study was to clarify the mechanism of resistance of F. fujikuroi to phenamacril and the fitness of resistant strains. RESULTS: The nine F. fujikuroi strains examined were highly resistant to phenamacril. Eight of them had the point mutation TCA (Ser) → CCA (Pro) at codon 219 in the Myosin-5 protein, while the other had the point mutation TCA (Ser) → TTA (Leu) at codon 219. Myosin-5 replacement between resistant and sensitive strains confirmed that the point mutation in Myosin-5 caused the resistance of F. fujikuroi to phenamacril. Docking of phenamacril into the modeled binding pocket of Myosin-5 showed that the affinity between phenamacril and Myosin-5 decreased and a hydrogen bond could not be formed between phenamacril and the amino acid at codon 219 after it changed to Pro or Leu. There was no cross-resistance between phenamacril and other fungicides. The eight resistant strains containing the point mutation S219P had almost the same fitness as the sensitive strains, while the one resistant strain containing the point mutation S219 L showed decreased mycelial growth, sporulation and pathogenicity. CONCLUSION: In the field, the point mutation S219P or S219 L in Myosin-5 conferred high resistance to phenamacril in F. fujikuroi. The point mutation S219P did not affect the fitness of F. fujikuroi, while the point mutation S219 L decreased its fitness. © 2017 Society of Chemical Industry.