Simultaneous detection of thirteen exons of dystrophin gene by optimized multiplex PCR assay to screen Duchenne/Becker muscular dystrophy

Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA)signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3–0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics

A study of feto-maternal outcome in case of premature rupture of membrane at a tertiary care center

Background: Premature rupture of membranes is the rupture of the fetal membranes in the absence of uterine contraction or before the onset of labor. When this occurs before 37 weeks of gestation, it is termed as preterm premature rupture of membranes. Management depends upon gestational age and the presence of complicating factors. An accurate assessment of gestational age and knowledge of the maternal, fetal and neonatal risks are essential to appropriate evaluation, counselling, and care of patients with PROM. The purpose of the study is timely diagnosis and appropriate management of the cases of PROM and PPROM to improve maternal and neonatal outcomes. Methods: A Prospective study was performed at the department of obstetrics and gynecology, at a tertiary care center from August 2020 to December 2021. A clinical data sheet was made for recording all information about the pregnant women after taking their consent. And their maternal and neonatal outcomes were recorded. Results: a total of 150 cases of PROM and PPROM were taken during our study out of which 53.33% belong to the younger age group, 43.33% were primi gravida, 66.66% belonged to the lower socioeconomic class, 25.33% had a previous history of abortion followed by dilatation and evacuation, rate of cesarean delivery was 34.66% and rate of NICU admission of neonates was 15.78% and 57.87% babies had low birth weight and rate of stillbirth was 1.97%. whereas 34.66% of cases had various complications related to PROM. Conclusions: Individualized management of PROM cases depending on the gestational age and risk of complications and antibiotic coverage is the best way to achieve a good fetomaternal outcome

Simultaneous detection of thirteen exons of dystrophin gene by optimized multiplex PCR assay to screen Duchenne/Becker muscular dystrophy

31-42Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA) signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 μL PCR reaction includes primer(s) (0.3–0.5 μM each), dNTP mixture (160 μM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 μL), DNA template (250 ng/50 μL), BSA (0.4 μg/μL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics

Isolation and characterization of undescribed flavonoid from Abrus precatorius L. based on HPTLC-DPPH bioautography and its cytotoxicity evaluation

Abstract Background Naturally derived compounds play a tremendous role as a drug as well as lead structure for the development of APIs. Therefore, isolation and characterization of compounds from nature are needed to alleviate life-threatening diseases. A. precatorius L. belongs to the family Leguminosae and is valued for its medicinal properties. Therefore, in this study, efforts are being made to isolate bioactive entity based on HPTLC-DPPH bioautography from APHA extract. Among all the separated compounds on TLC plate, the one (APSP-3) at R f = 0.67 showed significant antioxidant activity, and hence, APSP-3 was further subjected to isolation, purification, and structural characterization using diverse analytical modus operandi such as 1D and 2D NMR, FTIR, HPLC–MS/MS, and elemental analysis. In addition, antioxidant and cytotoxicity evaluation of APHA extract and APSP-3 was pursued by standard DPPH and colorimetric MTT assays, respectively. Results Antioxidative isolated compound APSP-3 was scrutinized based on HPTLC-DPPH bioautography. The APSP-3 was found novel and spectroscopic data revealed the plausible structure; 7-hydroxy-3,5-dimethoxy-2-(4-((3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy) phenyl)-4H-chromen-4-one. Moreover, APSP-3 ascribed higher free radical scavenging activity with IC50 = 38.70 ± 3.5 µg/mL than standard ascorbic acid (75.19 ± 1.5 µg/mL). Cytotoxicity evaluation of APHA extract exhibited IC50 value 122.09 µg/mL for HepG2, 122.61 µg/mL for MCF-7, and 48.08 µg/mL for HCT116 cell lines, while APSP-3 displayed IC50 values 96.75 for HepG2, 61.67 for MCF-7, and 47.61 µg/mL for HCT116 cell lines. Conclusions In a nutshell, HPTLC-directed bioautography leads to the capturing of new flavonoid entity having antioxidant potency from APHA extract. The IC50 values obtained from cytotoxicity establish a dose–response relationship helping to determine the concentration at which a substance begins to exhibit toxic effects. This fundamental information is crucial for establishing safe dosage level in medical and pharmaceutical applications. Further, research engrossed in assessing other bioactivities involving in silico and in vivo studies obliged to offer a promising and secure portrayal for clinical implications. Graphical abstrac