Development of an optimized RT-LAMP test for the detection of SARS-CoV-2.

SARS-COV-2 is the causative agent of an acute respiratory syndrome called Coronavirus disease 2019 (COVID-19) with a varying mortality rate from 2019 to 2022. There are several measures for control and prevention of Covid-19 including using mask, vaccine injections, as well as screening the potential cases. We aimed to design and develop a molecular method (RT-LAMP) for detecting coronavirus in biological samples that is cheaper, faster and easier than conventional molecular methods. In this study, various reaction components were explored to make the optimal combination of an RT-LAMP master mix composition. The results revealed the ability of this RT-LAMP test in specifically identifying 100 copies of mixture of N and E genes in just 30-45 min. This study demonstrated the reliable performance of the RT-LAMP method for the detection of SARS-COV-2 in biological samples. Given the significant advantages of this method compared to the gold standard qRT-PCR, it can be employed as a promising tool for the diagnosis of coronavirus as well as other pathogenic viruses.

Cryopreservation of Iranian Markhoz goat fibroblast cells as an endangered national genetic resource.

BACKGROUND: The continuous accessibility of local animals for sustainable use is being eroded annually. Thus, a strategic vision for the conservation of biodiversity is of far-reaching emphasis to deal with unprecedented challenges in the local population extension facing in the future. This study aimed to establish and cryopreserve endangered Markhoz goat (Capra hircus) fibroblast cell lines in vitro. METHODS AND RESULTS: These primary fibroblast cells were isolated from 58 Iranian Markhoz goats and individually cultured by explant technique in DMEM medium supplemented with 10% FBS and 2 mM L-Glutamine, in the presence of Penicillin (200 U/ml)-Streptomycin (200 mg/ml) during the first passage number. The extracted cell lines were confirmed morphologically as fibroblast cells. The population doubling time for DMEM-cultured cells was 23 ± 0.5 h. Chromosomal analysis indicated a total chromosome number of 2n = 60 with > 95% frequency. The cultured cells were checked for bacteria, fungi, yeast, and mycoplasma contaminations and the results were reported negative. The efficiencies of the fluorescent protein encoded by VSV-G (pMDG) and lentiviral pCSGW vectors reported in a range of 65% value. According to the species identification analysis, the goat cell lines were banked and confirmed without any miss- and cross-contamination. CONCLUSIONS: The significant issue in this paper can be concluded about the first report of the establishment of endangered Markhoz goat cell banking inside the country. This study demonstrated the successful establishment of a genetically stable fibroblast bank as a valuable genetic resource for the endangered Iranian Markhoz goat breed.

Establishment and characterization of a PCOS and a normal human granulosa cell line.

Oocyte maturation is an important phase in fertility and any disorder in this process could lead to infertility. The most common disorder during folliculogenesis is polycystic ovary syndrome (PCOS). Due to the secretive activity of granulosa cells (GCs), they play a vital role in folliculogenesis. Although scientists use various cellular and molecular methods to have a better understanding of the mechanism of these cells, some limitations still exist in GC culture such as low primary cell yield and proliferation capability. Therefore, immortalization of primary cells is an approach to overcome these limitations. In the current study, GCs were obtained from two females, one with PCOS and one with normal folliculogenesis. In the first stage, we established two human GC (hGC) lines by immortalizing them through retrovirus-mediated transfer of the human telomerase reverse transcriptase (hTERT) and c-Myc genes. Subsequently, the normal and PCOS cell lines were characterized and were investigated for their growth features. The cell lines were also examined in terms of immortal markers of hTERT, follicle stimulating hormone receptor (FSHR), aromatase, anti-Müllerian hormone (AMH), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), estrogen, and progesterone. Our results indicated that the normal and PCOS cell lines both showed similar characteristics to GCs during the follicular stage in normal and PCOS women. The normal and PCOS cell lines demonstrate molecular mechanisms similar to that of GCs such as folliculogenesis, oogenesis, and steroidogenesis, which enable researchers to perform further investigations in future.

Identification of Bactrian camel cell lines using genetic markers.

Iranian Bactrian camel population is less than 100 animals. Iranian biological resource center produced more than 50 Bactrian camel fibroblast cell lines as a somatic cell bank for conservation animal genetic resources. We compared two type markers performance, including 14 random amplified polymorphic DNA (RAPDs) (dominant) and eight microsatellite (co-dominant) for cell line identification, individual identification and investigation genetic structure of these samples. Based on clarity, polymorphism, and repeatability, four RAPD primers were selected for future analysis. Four RAPD primers and eight microsatellite markers have generated a total of 21 fragments and 45 alleles, respectively. RAPD primers revealed fragment size between 150 to 2000 bp and gene diversity since 0.27 (IBRD) to 0.46 (GC10), with an average of 0.37. Microsatellite markers generated number of alleles per locus ranged from 3 to 11, with an average of 5.62 alleles. The observed heterozygosity ranged from 0.359 (IBRC02) to 0.978 (YWLL08), and expected heterozygosity ranged from 0.449 (IBRC02) to 0.879 (YWLL08). Bottleneck analysis and curve showed that Bactrian camel population did not experience a low diversity. RAPD profiles were especially suitable for investigation population genetics. All primers generated novel and polymorphic fragments. Briefly, our results show that a multiplex PCR based on these markers can still be valuable and suitable for authentication of cell lines, investigating gene diversity and conservation genetic resources in Bactrian camel, while new technologies are continuously developed.

Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.