Direct PCR amplification from saliva sample using non-direct multiplex STR kits for forensic DNA typing

Due to its proficiency to provide the most discriminating results for forensic applications, medical research and anthropological studies, multiplex PCR based STR analysis has been established as the most efficient technique in the forensic DNA analysis. Several multiplex amplification kits based on 4, 5 and 6 dyes chemistry are commercially available and used in forensic DNA typing across the globe. These multiplex PCR systems are routinely used for amplification of multiple STR loci (Autosomal, Y and/or X STR’s) in the DNA extracted from various biological samples. In the routine forensic DNA testing, DNA profile obtained is compared with the DNA profile of the reference sample, which takes a certain turnaround time and employs costly lab resources. Successive development in forensic DNA typing have resulted in advent of improved multiplex kits which have reduced the effective analysis time, cost and minimized the number of steps required in comparison to conventional forensic DNA typing. Specialized direct amplification compatible multiplex kits are also available nowadays. These kits are relatively costlier but still require few pre-processing steps, which does not make them worth the hefty cost. Herein, this study, we have used non-direct multiplex STR kits to assess their efficacy for direct amplification. In the present study, 103 saliva samples were directly amplified without any pre-treatment of the samples using thirteen non-direct multiplex kits (4 dyes, 5 dyes and 6 dyes chemistry based) for forensic DNA typing. Here, we report a validated direct PCR amplification protocol from the reference saliva samples by omitting DNA extraction and quantification steps, which resulted in 80% reduction of the turnaround time. The developed protocol is cost effective, time efficient and it does not compromise with the quality of DNA profiles. To the best of our knowledge, this is the first report for direct amplification of DNA with the most commonly used non-direct multiplex STR kits without any pre-treatment of the sample. Complete DNA profiles matching all the essential quality parameters were obtained successfully from all the tested samples.

DEVELOPMENT OF THE METHOD OF DECOMPOSITION OF THE ELECTRICAL CIRCUIT BY MEANS OF ITS STRUCTURING ON THE BASIS OF THE PHYSICAL PRINCIPLE OF OPERATION MODEL

A method of decomposition of the electrical circuit is considered, which allows to obtain its systemic representation, which simplifies the identification of the principles and features of its functioning. On the example of an electrical circuit of a transistor radio receiver of direct amplification, the process of its structuring is shown according to the physical principle of action laid down in it, which allows its further decomposition. The method allows you to represent an electrical circuit in the form of a four-level model, which makes it possible to work with it for developers of different qualifications.

Accelerated RNA detection using tandem CRISPR nucleases

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

Sequencing of clinical samples reveals that adaptation keeps establishing during H7N9 virus infection in humans

The H7 subtype avian influenza viruses (AIV) have a much longer history and their adaptation through evolution pose continuous threat to humans 1. Since 2013 March, the novel reasserted H7N9 subtype have transmitted to humans through their repeated assertion in the poultry market. Through repeated transmission, H7N9 gradually became the second AIV subtype posing greater public health risk after H5N1 2,3. After infection, how the virus tunes its genome to adapt and evolve in humans remains unknown. Through direct amplification of H7N9 and high throughput (HT) sequencing of full genomes from the swabs and lower respiratory tract samples collected from infected patients in Shenzhen, China, we have analyzed the in vivo H7N9 mutations at the level of whole genomes and have compared with the genomes derived by in vitro cultures. These comparisons and frequency analysis against the H7N9 genomes in the public database, 40 amino acids were identified that play potential roles in virus adaptation during H7N9 infection in humans. Various synonymous mutations were also identified that might be crucial to H7N9 adaptation in humans. The mechanism of these mutations occurred in a single infection are discussed in this study.

Treponema pallidum tprII subfamily genes internal fragments sequencing

Background. The modern system of molecular typing of the Russian population of T. pallidum makes it possible to obtain results with a significant dominance of the 14d/f type, which determines the need to increase the differentiating ability of the applied methods of molecular typing of T. pallidum. Aim. Identification and analysis of nucleotide sequence variability of internal gene fragments of the tprII family of Russian T. pallidum subsp. pallidum strains. Material and methods. The study of internal variable fragments of genes of the tprII family was carried out among 240 clinical isolates of T. pallidum obtained from the Central (Kaluga Region, Moscow), North Caucasian (Stavropol Territory), Far East (Republic of Sakha), Volga (Chuvash Republic), Southern (Astrakhan Region) and Siberian (Novosibirsk and Omsk Regions, Republic of Tyva) federal districts in 20142020. The sequence of internal variable fragments of genes of the tprII family was determined using capillary sequencialng technology. Results. The primers allowing both direct amplification of the internal variable region of the tprII genes subfamily and correct sequencing of their internal regions have been proposed. It was found one SNP at positions 1340 of tprG gene. The polymorphism differs the reference Nichols strain from globally distributed Street 14 genogroup variants. Conclusion. The variability of tprII subfamily genes nucleotide sequences in modern Russian strains of T. pallidum subsp. pallidum is an additional fund to increase the efficiency of the modern T. pallidum molecular typing system.