ABSTRACT
Background: Dirofilaria immitis is a cosmopolitan zoonotic, vector-borne parasite of carnivorous animals causing dirofilariasis in human beings. Common commercial serodiagnostic tests for canine dirofilariasis usually lead to different results in their sensitivity and specificity. The present study reports development of recombinant DgK [rDgK] antigen of D. immitis for accurate immunodiagnosis of D. Immitis-infected dogs using indirect ELISA test
Methods: The rDgK coding sequence was successfully sequenced, codon optimized and cloned in pET-24a[+] expression vector and then expressed in Escherichia coli. The recombinant DgK was affinity purified using Ni[+2] - charged HiTrap chelating column, followed by testing in Western blotting and enzyme-linked immunosorbent assays [ELISA] with dog sera from a dirofilariasis endemic area. The performance of rDgK ELISA was evaluated using 60 sera collected from suspected dogs, while molecular technique was used as a reference test
Results: Sera from positive control D. immitis infection produced a strong IgG antibody response to rDgK both in ELISA and Western blotting tests. The sensitivity and specificity related to diagnostic potential of rDgK for ELISA were 92.5% and 87.5%, respectively. The results of rDgK ELISA showed a high agreement [0.764] with molecular identification
Conclusions: The findings revealed that the developed new rDgK antigen is sensitive and specific for immunodiagnosis of canine dirofilariasis using ELISA test
ABSTRACT
Non-invasive therapeutic methods have recently been used in medical sciences. Enzymes have shown high activity at very low concentrations in laboratories and pharmaceutical, enabling them to play crucial roles in different biological phenomena related to living organism, especially human medicine. Recently, using the therapeutic methods based on non-invasive approaches has been emphasized in medical society. Researchers have focused on producing medicines and tools reducing invasive procedures in medical. Collagenases are proteins which catalyze chemical processes and break the peptide bonds in collagen. Collagen may be generated more than the required amount or produced in unsuitable sites or may not degrade after a certain time. In such cases, using an injectable collagenase or its ointment can be helpful in collagen degradation. In both in vitro and in vivo tests, it has been revealed that collagenases have several therapeutic properties in wound healing, burns, nipple pain and some diseases including intervertebral disc herniation, keloid, cellulite, lipoma among others. This review describes the therapeutic application of collagenase in medical sciences and the process for its production using novel methods, paving the way for more effective and safe applications of collagenases.
ABSTRACT
Asthma is caused by the combination of different factors. Current concepts of asthma pathogenesis emphasize on gene-environment interactions. Mega-genome scanning projects revealed that different Single Nucleotide Polymorphisms [SNPs] are related to asthma susceptibility. rs7216389-T is one of them that is related to childhood asthma and its effect on childhood asthma severity has been proved in different nations, however no study has been performed in Eastern Mediterranean and Middle East countries yet. To perform population genetic studies, a rapid and high-throughput screening method is necessary. High-resolution melting analysis is a rapid, powerful and accurate method, which is suitable for this type of studies. Therefore, it has been decided to develop a high-resolution melting method for rs7216389 locus genotyping in Iranian asthmatic children. In the current study, a high-resolution melting analysis method based on SYBR Green-I was developed to check the frequency of rs7216389-T mutation in Iranian asthmatic children for the first time. Second and third classes of intercalating dyes are commonly used for high-resolution melting method. However, in this study, SYBR Green-I was used for rs7216389 locus genotyping for the first time. Our results for 60 samples showed that SYBR Green-I has good efficacy for rs7216389 locus genotyping through high-resolution melting method in comparison with PCR-RFLP and sequencing. Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, high-throughput and economic to study the rs7216389 locus in asthmatic children and it is applicable for other similar population genetic studies
ABSTRACT
Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 [human immunodeficiency virus] and HCV [hepatitis C virus], it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument. The current research recruits a multiplex nucleic acid sequence base amplification [NASBA] in order to simultaneously detect HIV-1 and HCV genomes in patients' plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples. A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used. A total of 40 samples of HIV-1 [20 samples] and HCV [20 samples] were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively. By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients' plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.
Subject(s)
Humans , Coinfection/diagnosis , Hepacivirus/isolation & purification , HIV/isolation & purification , HIV Infections/diagnosis , Hepatitis C/diagnosis , Self-Sustained Sequence Replication , ResearchABSTRACT
Because of the lack of an effective and economical control strategy against malaria [the most devastating infectious disease in developing countries] Transmission-Blocking Vaccines [TBVs] concept has been raised in recent years, promising a more efficient way to malaria control. TBVs aim at interfering and/or blocking pathogen development within the vector, halting transmission to non-infected vertebrate host. Aminopeptidase N [APN] is one of the most potent proteins in parasite development in Anopheles malaria vectors, which is strongly co-localized with human malaria parasites in the mosquito midgut epithelium. Therefore, Aminopeptidase N is one of the best choices for a new TBV. In this study for the first time we used 3'-RACE to amplify APN gene in Anopheles stephensi [An.stephensi], a major malaria vector in Iran, Indian subcontinent up to China by using different sets of primers including exon junction, conserved and specific region primers. Full length of APN was sequenced stepwise, which could be applied in designing a new regional TBV and act as an essential component of malaria elimination program in An. stephensi distribution areas. Primers design and method modification should be set up exactly in approach based amplifications. From results we came to this conclusion that that 3'-RACE could be applied to amplified key regions which are be-yond reach