Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting.

The prompt detection of human papillomavirus and discrimination of its genotypes by combining conventional methods in new molecular laboratories is essential to achieve the global call of eliminating cervical cancer. After predicting the melting temperature of an approximately 221 bp region of the L1 gene from different HPV genotypes by bioinformatics software, an innovative technique based on the nested- high resolution melting was designed with three approaches and using conventional PCR, qPCR, and diagnostic standards. HPV-positive samples identified by microarray along with diagnostic standards were evaluated by qPCR-HRM and discordant results were subjected to sequencing and analyzed in silico using reference types. In addition to screening for human papillomavirus, nested-qPCR-HRM is one of the modified HRM techniques which can discriminate some genotypes, including 6, 16, 18, 52, 59, 68 and 89. Despite the differences in diagnostic capabilities among HRM, microarray and sequencing, a number of similarities between HRM, and sequencing were diagnostically identified as the gold standard method. However, the bioinformatics analysis and melting temperature studies of the selected region in different HPV genotypes showed that it could be predicted. With numerous HPV genotypes and significant genetic diversity among them, determining the virus genotype is important. Therefore, our goal in this design was to use the specific molecular techniques with several specific primers to increase sensitivity and specificity for discriminating a wide range of HPV genotypes. This approach led to new findings to evaluate the ability of different approaches and procedures in accordance with bioinformatics.

Molecular Evaluation of the Novel Coronavirus Infection of Cockroaches and Flies Collected from Kamkar-Arabnia Hospital in Qom City, Central Iran: With Innovated Internal Control.

Background: Due to the confirmation of the presence of the novel coronavirus in the feces and municipal sewerage system, and the feeding of domestic insects from fecal matter, as well as the ability of these insects to mechanically transmit microbes from the sewerage system. This study was aimed at molecular evaluation of the novel coronavirus infection isolated on cockroaches and flies collected from Kamkar-Arabnia Hospital in Qom City, Iran. Methods: Totally, 18 samples; (12 samples cockroaches and 6 flies) from the external surface of cockroaches and houseflies as well as their digestive system were prepared. After designed and synthetized exogenous heterologous internal control, the RNA was extracted to investigate the contamination of these samples with the novel coronavirus. To detect the virus, the E and RdRp genes were identified. Results: Investigation of coronavirus E gene using the multiplex one-step qPCR technique on the collected samples showed an amplification plot in CT= 35.70 related to the internal surfaces of cockroaches collected from the treatment and sick room of the hospital. Also, the design of internal control to ensure the accuracy of the extraction process was successful. Conclusion: According to the findings of the present study regarding detecting the presence of the coronavirus infection in the digestive system of domestic insects such as American cockroaches and considering their ability to mechanically transmit viruses, it is recommended to control the domestic insects that are in close contact with humans in crowded places such as hospitals and health centers during the Covid-19 pandemic.

Assessing genetic evolution and detecting human papillomavirus by matching two complementary highly sensitive approaches, nested-qPCR and sequencing.

Becoming armed with an appropriate strategy to isolate the minimum number of human papillomaviruses (HPV), regardless of DNA extraction method, can be a huge step in preventing false negative; it has a significant effect on the management and control of HPV infection among women's population. This study was conducted in Qom province, considering the risk factors associated with HPV. It was able to analyze genetic evolution in its genotypes and evaluated the limit of detection by a new diagnostic approach. Totally, 486 Pap smear samples were tested; then, the HPV DNA was developed by a semi-nested quantification PCR. Positive samples were sequenced and submitted to the GenBank (MG825048-MG825061). After alignment, phylogenetic and polymorphism analyses were performed on the sequenced samples with a number of GenBank sequences. The overall HPV prevalence among all women in Qom was 11.7%. HPV6 (43.24%) and HPV16 (6.75%) were the most frequent LR and HR genotypes, respectively. Although the Tajima's D of all genotypes was positive, it was negative individually. The position of genotypes 6, 11, and 73 was controversial on phylogenetic trees. Limit of detection (LOD) was obtained as about 10-100 copies per reaction in various genotypes of HPV by semi-nested qPCR. The nature of HPV could be preserved during natural selection. This research, through innovative usage of the primers, could detect different genotypes of the HPV, and inform the women society of the probable risk through its prevalence determination.