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Mycoviruses are viruses that naturally infect and replicate in fungi. They are widespread in all major fungal groups including plant and animal pathogenic fungi. Several dsRNA mycoviruses have been reported in Aspergillus fumigatus. Multiplex polymerase chain reaction (PCR) amplification is a version of PCR that enables amplification of different targets simultaneously. This technique has been widely used for detection and differentiation of viruses especially plant viruses such as those which infect tobacco, potato and garlic. For rapid detection, multiplex RT-PCR was developed to screen new isolates for the presence of A. fumigatus mycoviruses. Aspergillus fumigatus chrysovirus (AfuCV), Aspergillus fumigatus partitivirus (AfuPV-1), and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) dsRNAs were amplified in separate reactions using a mixture of multiplex primer pairs. It was demonstrated that in the presence of a single infection, primer pair mixtures only amplify the corresponding single virus infection. Mixed infections using dual or triple combinations of dsRNA viruses were also amplified simultaneously using multiplex RT-PCR. Up until now, methods for the rapid detection of Aspergillus mycoviruses have been restricted to small scale dsRNA extraction approaches which are laborious and for large numbers of samples not as sensitive as RT-PCR. The multiplex RT-PCR assay developed here will be useful for studies on determining the incidence of A. fumigatus mycoviruses. This is the first report on multiplex detection of A. fumigatus mycoviruses.

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The present study was undertaken to explore whether prodynorphin (PDYN) polymorphisms have an effect on the intensity of depressive symptoms and negative craving in heroin addicts in a sample of 100 heroin addicts and 108 controls. PDYN rs2281285, rs2225749 and rs910080 polymorphisms were analyzed by PCR-RFLP. Craving and the intensity of depressive symptoms were measured by the Substance Craving Scale and the Beck Depression Inventory-II, respectively. A significant association between depression severity and PDYN rs2281285 (P=0.026) and rs2225749 (P=0.038) polymorphisms was detected. PDYN rs2225749 variation showed a trend association with increased negative craving (P=0.066). We also examined the associations between heroin dependence and PDYN rs2281285, rs2225749 and rs910080 gene polymorphisms at the gene and haplotype levels. The AAA haplotype was more frequent in heroin addicts and shown to be significantly associated with increased risk for heroin dependence (OR, 8.922; 95% CI, 1.116-71.313; P<0.05). PDYN rs2281285 and rs2225749 variations affected the intensity of depressive symptoms, and PDYN rs2225749 polymorphism may contribute to the induction of negative craving in heroin addicts. Haplotype analysis revealed for the first time that addicts with the AAA haplotype of PDYN gene may be more prone to heroin dependence.

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Objective: Multiple environmental and genetic factors contribute to the progression of alcohol use disorder (AUD). Ghrelin is one of the important elements of the brain-gut axis that has been believed to involve in the pathophysiology of addiction. This study aimed to determine whether the GHRL Leu72Met gene polymorphism has an effect on the plasma acylated ghrelin levels in alcohol addicts for the first time. Method: A sample of 50 alcohol-dependent men and 50 controls were enrolled in this study. Acylated ghrelin levels were detected by ELISA kit. The GHRL Leu72Met polymorphism was analyzed by the standard PCR-RFLP method. Results: Acylated ghrelin levels were significantly higher in AUD patients than in controls, and were lower in AUD patients with Leu72Leu than those with Leu72Met and Met72Met. After detoxification, a dramatic decrease was seen in AUD patients having Leu72Met+Met72Met. The presence of 72Met allele was also found to be associated with an increased risk of AUD in Turkish men. Conclusion: It was indicated for the first time that the GHRL Leu72Met variant was associated with higher plasma acylated levels in patients with AUD. The GHRL Leu72 allele compared to the Met72 allele seemed to be protective against AUD in Turkish men. Taken together, despite the small number of subjects evaluated, the findings in this study suggested the effect of the GHRL Leu72Met polymorphism on plasma acylated ghrelin levels and alcohol addiction.

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