Validation Study of 6 Dye-Based SF25TM PCR Amplification Kit

Ever since the inception of STR-based forensic DNA analysis for human Identification and DNA profiling, the approach developed continuously. Most of these developments are pertaining to achieve the best polymorphic loci set for analysis, high inhibitor tolerance capability, increase in sensitivity, reduction in turnaround time, and cost-effectiveness. On the similar line, the SF25TM PCR amplification kit, a 6 fluorescent dye-chemistry based autosomal STR kit was developed, which analyses 25 STR loci viz., FAM fluorescent dye-labelled D3S1358, D13S317, D7S820, D16S539, D1S1656, and Penta E; HEX fluorescent dye-labelled TPOX, TH01, D2S1338, CSF1PO, and Penta D; SUM fluorescent dye-labeled D19S433, vWA, D21S11, D18S51, and D6S1043; LYN fluorescent dye-labelled Amelogenin, D8S1179, D5S818, D12S391, and FGA; PUR fluorescent dye-labelled D22S1045, Y indel; D2S441 and D10S1248, The developmental validation of the SF25 PCR Amplification reagents were carried out in this study to analyze its specificity, sensitivity, and concordance. This multiplex kit showed higher sensitivity with 125pg of DNA template generating a complete profile (with 100% allele call). Out of 83 samples, 10 samples showed aberrant triallelic pattern/aberrant heterozygous pattern at D16S539 locus. The possible discrepant homozygous condition at locus D1S1656 might be due to the invasion of alleles at D1S1656 locus present adjacent to D16S539 locus. Such invasion resulted due to the shortening of the allelic bin at the D1S1656 locus. The allelic ladder of the SF25TM PCR amplification kit reveals the allelic range of D1S1656 from 11 to 19.3 which can be interpreted according to the casework as deemed by the end-users for their samples.

A Unique Allele Variant at STR Locus D2S1338 in a Paternity Testing Case

Background: The relationship testing through DNA profiling may undesirably be affected by the rare allele variants, tri-allelic pattern and null alleles. Therefore, it is vital to report such anomalies. We report a paternity testing in a sexual assault case studied at Punjab Forensic Science Agency, Lahore Pakistan showing a unique allele variant in mother and child. Methods: DNA was extracted from the buccal swabs of reference samples using organic extraction method and DNA profiling was done for 15 autosomal STRs and amelogenin using Identifiler Plus kit. Results: A novel out of marker range (OMR) allele variant between STR Loci D16S539 and D2S1338 was observed in the DNA profiles of victim (mother) as well as the child. At STR locus D2S1338 Twenty one different allele variant are listed at STRBase ranging from 11 to 28. The allele variant observed in this case study was appeared at less than marker range (< D2S1338) with a size of 297.50 bp. The novel variant OMR allele at D2S1338 was labeled as allele 13, when compared to the other allele in allelic ladder. Moreover, the PFSA DNA database was searched for this unique allelic variation and it was found that this was present in only two other samples of distinct cases. Conclusion: The overall frequency of this unique allele variant was 3 in 10,125 unrelated individuals with frequency of occurrence of 0.0296. According to our limited knowledge it is the first report of a novel OMR allele variant at D2S1338 in Pakistani Population.

A Technology for Genetic Identification of Varieties and Wild Forms of Grapes Based on Multilocus Microsatellite Analysis

The genotyping technology has been developed based on 9 microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VVMD28, VVMD25, VVMD32, VrZAG62, VrZAG79). It can be used for efficient, accurate and fast identification of grape varieties and forms (genus Vitis). The proposed approach includes a multiplex PCR of all loci followed by electrophoretic analysis of DNA fragments in one capillary of a genetic analyzer. The application of an additional length standard, an allelic ladder, consisting of all possible DNA fragments of the analyzed microsatellite loci, is one of the key features of the technology, which ensures the accuracy and reproducibility of the results. The advantage of the proposed technology is the ability to standardize and automate the procedure using 96-well plates, which opens up the possibility of conducting mass analyses. As a result of the study of varieties and forms of Vitis genus species, genetic passports were obtained, according to which a dendrogram was constructed, reflecting the genetic relationship of the studied samples. The developed technology makes it possible to distinguish varieties and wild forms of grapes; it can be used for their identification and determination of genetic distance between them, as well as for assessment of planting material and protection of breeders' rights. Vitis, grapes, microsatellites, SSR, genotyping, multiplex PCR, DNA fragment analysis The work was carried out within the framework of the state assignment on the topic "Development of New Technologies for Genetic Analysis of Forms of Agricultural Plants to Accelerate and Control the Selection Process" (project no. 0574-2019-0003).

A reference allelic ladder for Western Capercaillie (Tetrao urogallus) and Black Grouse (Tetrao tetrix) enables linking grouse genetic data across studies

Rapid anthropogenic climate change and progressing habitat degradation are considered top threats to biodiversity. The employment of demanding umbrella species as indicators for ecosystem health is a popular and cost-effective strategy that facilitates continuous monitoring and evaluation within a long-term conservation management scheme. The Western Capercaillie (Tetrao urogallus) and the Black Grouse (Tetrao tetrix) are both considered viable candidates due to their extensive habitat requirements, the possibility for conservative, non-invasive sampling, and their broad popular appeal. Regional population surveys based on genetic data from Short Sequence Repeat (SSR) analysis are being conducted throughout the Palearctic. However, to ensure reliable comparability among laboratories, standardization is required. Here, we report a catalogue of fifty fully characterized reference alleles from twelve SSR loci and the construction of a customizable allelic ladder for genotyping and individualization in Western Capercaillie and Black Grouse. This methodological improvement will help to cost-efficiently generate and collate supraregional data from different grouse surveys and thereby contribute to conservation management. Reference alleles and ladders can be obtained on demand.

Molecular Study about some Genes among Salmonella Typhi Isolates un Hilla / Iraq

In this study, (100) blood sample were collected from patient suspected with typhoid fever how were visits the Alhashimiya, Hilla Teaching Hospital and Morjan Teaching Hospital in Babylon province (Iraq) during the period November (2018) to February (2019). Identification was done by cultural and biochemical tests, and finally identification by Vitek2. Results showed that positive were (16) isolates of Salmonella enteric serovar typhi by Vitek2 (16%) from the(100) blood. Molecular study was accomplished first by the isolation of DNA from Salmonella typhi isolates, they used method had succeeded in extraction of genomic DNA from all isolates. Second by using PCR technique to detection four gene by using a specific PCR primer were done by comparison with allelic ladder which gave a (578bp) it was found that (flicA) gene present in (15) isolates (93.75%) of the positive sample. viaB gene was also detected in S. typhi sample and found that in (15) isolates (93.75%) of the positive sample which gave molecular length (438bp) band. Molecular detection of sipA gene in S. typhi present of this gene in all (16) isolated (100%) positive, which gave molecular length (1126bp). Molecular detection of sdiA gene was done for (16) isolated of S. typhi, and the results showed that (15) isolates (93.75%) the positive results for sdiA virulence were detected by the presence of (274bp) band compared with allelic ladder. This study was conducted to identify the presence of Salmonella enterica serovar typhi within five isolated bacterial samples. One genetic locus covering a particular coding portion within the sdiA gene, which is responsible on the encoding of cell-division regulatory protein, was selected for amplification

Y-profile evidence: close paternal relatives and mixtures

We recently introduced a new approach to the evaluation of weight of evidence (WoE) for Y-chromosome profiles. Rather than attempting to calculate match probabilities, which is particularly problematic for modern Y-profiles with high mutation rates, we proposed using simulation to describe the distribution of the number of males in the population with a matching Y-profile, both the unconditional distribution and conditional on a database frequency of the profile. Here we further validate the new approach by showing that our results are robust to assumptions about the allelic ladder and the founder haplotypes, and we extend the approach in two important directions. Firstly, forensic databases are not the only source of background data relevant to the evaluation of Y-profile evidence: in many cases the Y-profiles of one or more relatives of the accused are also available. To date it has been unclear how to use this additional information, but in our simulation-based approach its effect is readily incorporated. We describe this approach and illustrate how the WoE that a man was the source of an observed Y-profile changes when the Y-profiles of some of his male-line relatives are also available. Secondly, we extend our new approach to mixtures of Y-profiles from two or more males. Surprisingly, our simulation-based approach reveals that observing a 2-male mixture that includes an alleged contributor’s profile is almost as strong evidence as observing a matching single-contributor evidence sample, and even 3-male and 4-male mixtures are only slightly weaker.

Genotypic detection of some virulence factors among Aeromonas hydrophila Isolated from Diarrhea Cases (Iraq)

The present study included the detection ofsome virulence factorsof Aeromonas hydrophila under molecular level to clinical isolates were taken from patients suffering from diarrhea during the period from July (2017) to October (2017). Molecular detection of Hemolysin gene (ahh) was done for all isolates. The results showed that all isolates (100%) gave positive results for this virulence gene. the positive results were detected by the presence of (130) bp bands when compared with allelic ladder. The genomic DNA of the samples was extracted and bands were observed by performing agarose gel electrophoresis. When PCR was performed,results clearly indicate that all isolated organisms contained serine protease gene and all the amplified products produced a band at the level of (900 bp) when compared with the allelic ladder. Molecular detection of this gene was carried out by using a specific PCR primer were done by comparison with allelic ladder which gave a (309bp) It was found that (Aerolysin) gene present in (12) (75%) of the positive samples. Lip gene was also detected in A. hydrophila samples and found that all 16 samples (100%) gave positive results to this gene which gave molecular length (382) bp. Molecular study was carried out to show the sequence identity of cytotonic enterotoxins gene in Aeromonas spp. to that in A. hydrophila. Analysis of the A. hydrophila genome revealed a number of a putative virulence factors,including a gene that heat-labile cytotonic enterotoxin (alt). Our study showed that all (16) isolates (100%) gave positive results to this gene,which gave molecular length (442)bp. Molecular detection of cytotonic enterotoxins gene (ast) was done for (16) A. hydrophila isolates and the results showed that all isolates have this gene (100%). The positive results for (ast) virulence were detected by the presence of (331) bp band compared with allelic ladder.