Background: The invention of Polymerase Chain Reaction (PCR) marked a breakthrough in biomedical research. Its invention divided the timeline into an era before and after PCR. Because of its multiple applications, it has become a vital tool for clinical researchers and diagnosticians. A effective PCR experiment requires adequate knowledge of each reaction component and stepby- step procedure to attain the optimized results. This research aims to employ optimization strategies that are easy to perform, cost-effective, and do not require PCR kits for the generation of amplicons for TYR, MITF, and SOX10 genes and can be used in sequence analysis. Materials and Methods: Whole blood samples were used to extract genomic DNA with an inorganic method. DNA quantification was done by spectrophotometry analysis. Optimization strategies were adopted to generate PCR products of candidate genes and visualized by agarose gel electrophoresis. Sanger sequencing was performed to check the quality and specificity of generated amplicons via optimization strategies. Results: This study demonstrates a novel approach for troubleshooting failed reactions without the use of PCR kits. The result indicated that use of sterilized material and reagents along with optimum MgCl2 concentration (2.0-3.0 mM), DNA quantity (25 ng/μL), and annealing temperature (54-60°C) are necessary to achieve successful amplification. Sequence analysis revealed no background noise often associated with sequencing results. Conclusion: Optimizing MgCl2 concentration, DNA quantity, annealing temperature, along with the use of contamination-free material and reagents are essential steps in PCR optimization. Following this guide, anyone lacking proper supervision, and with little or no knowledge of the procedure should attain the desired results.

Livestock is an important commodity playing a major role in the global economy. Red meat plays an important role in human life, as it is a good source of animal protein and energy. The fatty acid content of beef has been shown to impact the eating experience and nutritional value of beef. Therefore, this study aimed to identify genomic regions which can account for genetic variation in meat fatty acid content. Genotypes imputed to the Illumina BovineHD 770K BeadChip were used in this study. Thirty-six 1-Mb genomic regions with a posterior probability of inclusion (PPI) greater than 0.90 were identified to be associated with variation in the content of at least one fatty acid. The genomic regions (1Mb) which were associated with more than one fatty acid trait with high genetic variance and harbored good candidate genes were on Chromosome (Chr) 6 (fatty acid binding protein 2), Chr 19 (thyroid hormone receptor alpha, fatty acid synthase), Chr 26 (stearoyl-CoA desaturase), and Chr 29 (thyroid hormone responsive, fatty acid desaturase 2, and fatty acid desaturase 3). Further studies are required to identify the causal variants within the identified genomic regions. Findings from the present study will help to increase understanding of the variation in fatty acid content of beef and help to enhance selection for beef with improved fatty acid composition.

Hepatitis C virus (HCV) is a major threat to public health being the leading cause of chronic liver disease with 92 to 149 million people affected worldwide. A rapid and accurate diagnosis is a key to prevent viral transmission and the management of disease progression. Here we describe a hand-held device for rapid detection of HCV infection called as STALLION (Single Tube Analysis using LAMP, LED and ION-sensing). A custom detection probe was designed to dip into a PCR tube. The probe comprises an ion sensitive field effect transistor (ISFET), a micro-capillary based Ag/AgCl reference electrode and a temperature sensor. In vitro replication of HCV genomic RNA was performed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detected in real-time by ISFET bio-sensing of released H⁺ ions. Incubation at 60 °C was provided by a novel low-cost concept based on heat dissipated from power LED(s). With this system, HCV positive samples with 10¹, 10², 10³ copies per ml were detected in as less as 30, 18 and 13 minutes respectively compared to no-template control (NTC). The detection limit was comparable to those of available methods such as nested PCR. No significant false positive signal was observed for HCV negative samples. The results can be viewed on an accompanying LCD screen or alternatively can be transferred to a computer or smart phone. The STALLION system provides analysis in a conventional PCR tube format avoiding any complex fluidics or instrumentation requirements. Such a system will be particularly useful for rapid and reliable clinical diagnosis of HCV RNA by users with no prior expertise and provide the basis for point-of-care (POC) testing in limited resource settings especially for developing countries.

The pandemic SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has created a widespread panic across the globe especially in the developing countries like Pakistan. The lack of resources and technical staff are causing havoc challenges in the detection and prevention of this global outbreak. Therefore, a less expensive and massive screening of suspected individuals for COVID-19 is required. In this study, a user-friendly technique of reverse transcription-loop mediated isothermal amplification (RT-LAMP) was designed and validated to suggest a potential RT-qPCR alternate for rapid testing of COVID-19 suspected individuals. A total of 12 COVID-19 negative and 72 COVID-19 suspected individuals were analyzed. Both RT-qPCR and RT-LAMP assays were performed for all the individuals using open reading frame (ORF 1ab), nucleoprotein (N) and Spike (S) genes. All 12 specimens which were negative using RT-qPCR were also found negative using RT-LAMP assay. Overall 62 out of 72 positive samples (detected using RT-qPCR) were found COVID-19 positive using RT-LAMP assay. Interestingly all samples (45) having Ct values less than 30 showed 100% sensitivity. However, samples with weaker Ct values (i.e., => 35) showed 54% concordance, suggesting potential false negatives or false positives in RT-LAMP or RT-qPCR results, respectively. Overall comparative assessment showed that RT-LAMP assay showed strong sensitivity and specificity and can be used as an alternative strategy for rapid COVID-19 testing. Hence, based on fast processing time, minimal risk of specimens transfer and utilizing available resources, LAMP based detection of COVID-19 is strongly advocated especially for developing countries.

Congenital idiopathic nystagmus (CIN) is an oculomotor disorder characterized by repetitive and rapid involuntary movement of the eye that usually develops in the first six months after birth. Unlike other forms of nystagmus, CIN is widely associated with mutations in the FRMD7 gene. This study involves the molecular genetic analysis of a consanguineous Pakistani family with individuals suffering from CIN to undermine any potential pathogenic mutations. Blood samples were taken from affected and normal individuals of the family. Genomic DNA was extracted using an in-organic method. Whole Exome Sequencing (WES) and analysis were performed to find any mutations in the causative gene. To validate the existence and co-segregation of the FRMD7 gene variant found using WES, sanger sequencing was also carried out using primers that targeted all of the FRMD7 coding exons. Additionally, the pathogenicity of the identified variant was assessed using different bioinformatic tools. The WES results identified a novel nonsense mutation in the FRMD7 (c.443T>A; p. Leu148 *) gene in affected individuals from the Pakistani family, with CIN resulting in a premature termination codon, further resulting in the formation of a destabilized protein structure that was incomplete. Co-segregation analysis revealed that affected males are hemizygous for the mutated allele c.443T>A; p. Leu148 * and the affected mother is heterozygous. Overall, such molecular genetic studies expand our current knowledge of the mutations associated with the FRMD7 gene in Pakistani families with CIN and significantly enhance our understanding of the molecular mechanisms involved in genetic disorders.

One of the aggressive and lethal cancers, pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and resistance to conventional treatments. Moreover, the tumor immune microenvironment (TIME) plays a crucial role in the progression and therapeutic resistance of PDAC. It is associated with T-cell exhaustion, leading to the progressive loss of T-cell functions with an impaired ability to kill tumor cells. Therefore, this study employed single-cell RNA sequencing (scRNA-seq) analysis of a publicly available human PDAC dataset, with cells isolated from the primary tumor and adjacent normal tissues, identifying upregulated genes of T-cells and cancer cells in two groups (“cancer cells_vs_all-PDAC” and “cancer-PDAC_vs_all-normal”). Common and unique markers of cancer cells from both groups were identified. The Reactome pathways of cancer and T-cells were selected, while the genes implicated in those pathways were used to perform PPI analysis, revealing the hub genes of cancer and T-cells. The gene expression validation of cancer and T-cells hub-genes was performed using GEPIA2 and TISCH2, while the overall survival analysis of cancer cells hub-genes was performed using GEPIA2. Conclusively, this study unraveled 16 novel markers of cancer and T-cells, providing the groundwork for future research into the immune landscape of PDAC, particularly T-cell exhaustion. However, further clinical studies are needed to validate these novel markers as potential therapeutic targets in PDAC patients.