[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Molecular diagnosis of echinococcosis in patients based on frozen paraffin tissue samples and fixed formalin and hydatid cysts isolated from livestock in a slaughterhouse.

Background: Various genotypes of Echinococcus granulosus have been studied in high-disease-risk areas and identified as causative agents of cystic echinococcosis (CE). This study was performed to examine and identify the molecular hydatid cyst in the dissected human specimens in paraffin tissue, and the dissected animal cyst was characterized using the DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 1 (ITS1). Materials and Methods: To determine the molecular properties of E. granulosus, 20 hydatid cyst samples (including 6 sheep samples, 9 camel samples, and 10 human paraffin samples) were collected from Zahedan and Zabol cities. After DNA extraction, molecular PCR was performed, and RFLP was evaluated. In this study, the Taq1 endonuclease cleavage enzyme was used. Results: The patterns of DNA bands found in the isolates from human CE and animal bladder cysts were the same, as indicated by the results of ribosomal DNA-ITS1 amplification from E. granulosus. Two nested primer pairs were used. The rough size of the enhanced ITS1 piece was 444 and 391 base pairs (bp), individually. After cutting the PCR product with the Taq1 enzyme, the patterns of the fragments revealed that the samples had two identical RFLP patterns. The aftereffects of this study showed that the parasite genotypes confined to sheep, camels, and people had hereditary changes. Conclusion: The transcendent type of E. granulosus sensu lato in the area is E. granulosus sensu stricto, which featured the meaning of the sheep/canine cycle in human transmission. Albeit the band profile in the camel is now and again like the sheep strain, RLFP can be recognized utilizing the PCR strategy, and two differentiating band profiles using the chemical were found in this review.

Detection of Booroola Polymorphism of Bone Morphogenetic Protein Receptor 1b and Embrapa Polymorphism of Growth Differentiation Factor 9 in Sheep in Thailand.

This study aimed to investigate the appearance and frequencies of the Booroola polymorphism of the bone morphogenetic protein receptor 1b (BMPR1B) gene (FecB) and the Embrapa polymorphism of the growth differentiation factor 9 (GDF9) gene (FecGE) in sheep in Thailand. A total of 454 crossbred sheep blood samples were collected from four provinces in Thailand during August 2022 to July 2023. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the FecB and FecGE genotypes. The history of ewe birth types was collected from the owners to analyze the association between fecundity (Fec) genotypes and the history of birth types. The genotypic frequencies of FecB for homozygous genotype (B/B), heterozygous genotype (+/B), and wildtype (+/+) were 0.22%, 1.54%, and 98.24%, respectively. Meanwhile, the genotypic frequencies of FecGE for homozygous genotype (E/E), heterozygous genotype (+/E), and wildtype (+/+) were 0.00%, 2.42%, and 97.58%, respectively. Furthermore, three ewes exhibited both FecB and FecGE genotypes. Fisher's exact test revealed that possession of the FecB genotype was associated with multiple births (p < 0.01). Both FecB and FecGE mutations were identified in crossbred sheep in Thailand. Sheep containing FecB allele could be alternative candidates to be selected to improve the prolificacy of crossbred sheep in Thailand.

PCR-RFLP for Distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method for rapid distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex， so as to avoid the influence of genetic confusion on drug safety. MethodThe DSS-tagged sequences of Periplocae Cortex were obtained from the Chloroplast Genome Information Resource （CGIR） and analyzed to find the enzymatic cleavage sites that were different from those of Acanthopanacis Cortex and Lycii Cortex. The specific enzymatic cleavage site， Cla I， of Periplocae Cortex was selected， on the basis of which the primers for PCR-RFLP were designed. Furthermore， the factors such as annealing temperature， number of cycles， Taq enzyme， PCR instruments， and enzymatic treatment time that may influence PCR-RFLP were studied. The established PCR-RFLP method was applied to the identification of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex samples produced in different regions. ResultThe PCR-RFLP at the annealing temperature of 59 ℃ and with 40 cycles showed clear bands of the samples. When the enzyme digestion time was 30 min. The reaction produced the target bands at about 140 bp and 290 bp for both Periplocae Cortex and its original plant and only a band at about 430 bp for Acanthopanacis Cortex， Lycii Cortex， and their original plants. The method can accurately distinguish Periplocae Cortex from its confounders Acanthopanacis Cortex and Lycii Cortex. ConclusionThe PCR-RFLP method for distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex was established. It has high stability， sensitivity， and applicability， providing a reference for the quality control of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex.

A Rapid PCR-RFLP Method for Assessing Heterozygosity of Murraya paniculata Germplasm / 中国实验方剂学杂志

ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

Molecular Characterization of Haemoglobin E.

OBJECTIVE: To detect mutation in cases having haemoglobin A2 level >7% on high performance liquid chromatography. METHODS: The cross-sectional, descriptive study was conducted from July 2017 to December 2018 at the Department of Haematology and Human Genetics and Molecular Biology, University of Health Sciences, Lahore, Pakistan, and comprised patients of either gender with haemoglobin A2 ≥7%. The samples were collected from different cities of Punjab in collaboration with the Punjab Thalassemia Prevention Programme, Lahore. The samples were subjected to complete blood count and high performance liquid chromatography using automated haematology analysers and variant-II beta thalassemia short programme, respectively. To analyse haemoglobin E mutations at the molecular level, polymerase chain reaction-restriction fragment length polymorphism (PCR_RFLP) was performed using a type IIS restriction endonuclease known as Mnl1 (derived from Moraxella nonliquefaciens) to cleave DNA at specific sites and the results were further confirmed on randomly selected samples using Sanger sequencing. Data was analysed using SPSS 25. RESULTS: Of the 39 patients, 15(38.5%) were males and 24(61.5%) were females. The overall median age was 14 (23) years. There were 29 (74.4%) patients with thalassemia family history, and 22(56.4%) had a positive family history of transfusion related to thalassemia, while no patient had a family history of iron therapy. The median haemoglobin A, haemoglobin A2 and haemoglobin F levels were 72.2 (65.2-79.1) %, 26.6 (19.1-34.0) % and 0.9 (-0.8-2.6) %, respectively. After molecular investigation, HbAE mutation was found in 23(59%) patients, while wild type HbAA genotype was found in 16(41%). The heterozygous HbE mutation was present in 23(59%) patients. CONCLUSIONS: Frequently missed/undiagnosed cases of haemoglobin E that co-elute with haemoglobin A2 in the same high performance liquid chromatography window were detected among those with haemoglobin A2 ≥7%.

Prevalence and Clinical Characteristics of Mitochondrial DNA Mutations in Korean Patients With Sensorineural Hearing Loss.

BACKGROUND: Mutations in mitochondrial DNA (mtDNA) are associated with several genetic disorders, including sensorineural hearing loss. However, the prevalence of mtDNA mutations in a large cohort of Korean patients with hearing loss has not yet been investigated. Thus, this study aimed to investigate the frequency of mtDNA mutations in a cohort of with pre- or post-lingual hearing loss of varying severity. METHODS: A total of 711 Korean families involving 1,099 individuals were evaluated. Six mitochondrial variants associated with deafness (MTRNR1 m.1555A>G, MTTL1 m.3243A>G, MTCO1 m.7444G>A and m.7445A>G, and MTTS1 m.7471dupC and m.7511T>C) were screened using restriction fragment length polymorphism. The prevalence of the six variants was also analyzed in a large control dataset using whole-genome sequencing data from 4,534 Korean individuals with unknown hearing phenotype. RESULTS: Overall, 12 of the 711 (1.7%) patients with hearing loss had mtDNA variants, with 10 patients from independent families positive for the MTRNR1 m.1555A>G mutation and 2 patients positive for the MTCO1 m.7444G>A mutation. The clinical characteristics of patients with the mtDNA variants were characterized by post-lingual progressive hearing loss due to the m.1555A>G variant (9 of 472; 1.9%). In addition, 18/4,534 (0.4%) of the Korean population have mitochondrial variants associated with hearing loss, predominantly the m.1555A>G variant. CONCLUSION: A significant proportion of Korean patients with hearing loss is affected by the mtDNA variants, with the m.1555A>G variant being the most prevalent. These results clarify the genetic basis of hearing loss in the Korean population and emphasize the need for genetic testing for mtDNA variants.

Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes.

IMPORTANCE: In this study, comprehensive analysis of 82,237 global porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) open reading frame 5 sequences spanning from 1989 to 2021 refined PRRSV-2 genetic classification system, which defines 11 lineages and 21 sublineages and provides flexibility for growth if additional lineages, sublineages, or more granular classifications are needed in the future. Geographic distribution and temporal changes of PRRSV-2 were investigated in detail. This is a thorough study describing the molecular epidemiology of global PRRSV-2. In addition, the reference sequences based on the refined genetic classification system are made available to the public for future epidemiological and diagnostic applications worldwide. The data from this study will facilitate global standardization and application of PRRSV-2 genetic classification.

Changes in the Endophytic Bacterial Community of Brassica rapa after Application of Systemic Insecticides.

Insecticides not only control target pests but also adversely affect non-target communities including humans, animals, and microbial communities in host plants and soils. The effect of insecticides on non-target communities, especially endophytic bacterial communities, remains poorly understood. Two phases of treatments were conducted to compare the trends in endophytic bacterial response after insecticide application. Endophytic bacteria were isolated at 2 and 4 weeks after germination. Most insecticide treatments showed a declining trend in bacterial diversity and abundance, whereas an increasing trend was observed in the control. Therefore, insecticide use negatively affected non-target endophytic bacterial communities. Bacillus spp. was mostly dominant in the early stage in both insecticide treatment and control groups. Nevertheless, in the matured stage, mostly bacteria including Pseudomonas spp., Priestia spp. were dominant in groups treated with high insecticide concentrations. Therefore, plants can regulate and moderate their microbiome during their lifecycle depending on surrounding environmental conditions.

Simple confirmation methods for rare but impaired variants of human flavin-containing monooxygenase 3 (FMO3) found in an updated genome resource databank.

Forty-seven new nonsense or missense human flavin-containing monooxygenase 3 (FMO3) variants were recently identified in an updated Japanese population reference panel. Of these, 20 rare single-nucleotide substitutions resulted in moderately or severely impaired FMO3 activity. To easily identify these 20 FMO3 variants (2 stop codon mutations, 2 frameshifts, and 16 amino-acid substitutions) in the clinical setting, simple confirmation methods for impaired FMO3 variants are proposed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) or allele-specific PCR methods. Using PCR-RFLP, FMO3 variants p.Arg51Gly, p.Met66Lys, p.Asn80Lys, p.Val151Glu, p.Val187fsTer25, p.Gly193Arg, p.Val283Ala, p.Asp286His, p.Val382Ala, and p.Phe451Leu were digested by the designated restriction enzymes and confirmed using reference cDNAs. In contrast, the FMO3 variants p.Gly39Val, p.Arg238Ter, p.Arg387Cys, p.Arg387His, p.Leu457Trp, and p.Met497Arg were not digested, whereas the wild type was digested. FMO3 variants p.Gly11Asp, p.Lys416fsTer72, p.Gln427Ter, and p.Thr453Pro were confirmed using allele-specific PCR systems. The previously identified FMO3 p.Arg500Ter variant has a relatively high frequency and was differentiated from p.Arg500Gln in two steps, i.e., enzyme restriction followed by allele-specific PCR, similar to the method for p.Arg387Cys and p.Arg387His. These systems should facilitate easy detection in the clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearance possibly caused by impaired FMO3 function.

[Trichophyton tonsurans Infection].

Trichophyton tonsurans infection has been prevalent among individuals involved in contact sports in Japan since about 2000. The present review focuses on its diagnosis, molecular epidemiology, drug susceptibility, and infection control. The most commonly observed lesions of T. tonsurans, an anthropogenic dermatophyte, are tinea corporis and tinea capitis. However, the presence of asymptomatic carriers must be considered for infection control. Genotypic epidemiology using restriction fragment length polymorphisms (RFLP) in the non-transcribed spacer (NTS) region of the ribosomal RNA gene showed a lack of diversity of genotypes, and only the NTS I genotype is detected at present. In regard to drug susceptibility, terbinafine drug resistance has not been found to be associated with the RFLP genotypes, and it is assumed that there are no terbinafine-resistant strains in Japan. T. tonsurans coexisted with other fungi and bacteria in the scalp of asymptomatic carriers without affecting species diversity. T. tonsurans is an anthropogenic dermatophyte and may be difficult for the human immune system to eliminate. During an infection outbreak, screening of infection and treatment including asymptomatic carriers are essential to eradicate the infection.

Puke or poop? Comparison of regurgitate and faecal samples to infer alpine grasshopper (Paprides nitidus Hutton) diet in experimental plant communities.

Characterising plant-herbivore interactions is important to understanding the processes that influence community structure and ecosystem functioning. Traditional methods used to identify plant-herbivore interactions are being superseded by non-destructive molecular approaches that can infer interactions with greater resolution and accuracy from environmental DNA (e.g. faeces and regurgitate). However, few studies have compared the success of using different sample types and whether they provide similar or contrasting information about species' diet. Here we compared the success of DNA amplification and host plant species identification using restriction fragment length polymorphism (RFLP) applied to faecal and regurgitate samples collected from alpine grasshoppers Paprides nitidus Hutton during a grassland community mesocosm experiment. We found that DNA amplification success was 23% and 86% higher for faecal than regurgitate samples from female and male grasshoppers, respectively. In contrast, successful host plant identification using RFLP was 9% higher for regurgitate than faecal samples. The mean number of host plant species identified per sample (1.40) did not differ between sample types or grasshopper sexes. Of the 136 paired faecal-regurgitate samples, just 41% and 74% produced exactly or partially matching host plant identifications, respectively, indicating that different sample types provided complementary information about herbivore diet. Some plant species were more likely to be identified from faecal samples than expected by chance, and we found that this identification bias skewed towards plant species with higher investment in leaf tissue. We conclude that multiple sample types may be required to fully characterise an invertebrate herbivore species' diet.

Association of MMP7 T > C Gene Variant (rs10502001) and Expression in Chronic Obstructive Pulmonary Disease.

Patients with chronic obstructive pulmonary disease (COPD) have obstructed airflow through their lungs. Single nucleotide polymorphisms in matrix metalloproteinases (MMPs) genes and the risk of COPD have been the subject of numerous studies, with conflicting results. This investigation was conducted to determine whether the MMP7 (T>C) gene variant (rs10502001) was associated with an increased risk of COPD. A case-control study was conducted with 360 subjects (180 healthy controls and 180 COPD cases). The polymerase chain reaction (PCR)-restriction fragment length polymorphism method was used to genotype the SNP rs1050200. mRNA expression of MMP7 was performed using RT-PCR. The genotypic/allelic frequencies and carriage rates of rs10502001 (T>C) polymorphism were evaluated in 180 each of healthy controls and COPD cases. Cases have higher TC/CC genotype frequencies than controls. The "CC" genotype was found to be significantly associated with increased COPD risk (p = 0.016). The "C" allele frequency was higher in cases than in controls and showed significant association with COPD (p = 0.005). The carriage rate frequencies of T(-) and C(+) were significantly higher in cases than in controls (p = 0.031 and 0.047, respectively). MMP7 expression was significantly upregulated (p = 0.001) in COPD cases as compared with the controls. In addition, comparisons of MMP7 expression between the COPD cases with different genotypes showed that the "CC" genotype cases had significantly higher expression than those with "TT" genotype. The present findings showed statistically significant correlation of MMP7 (T>C) polymorphism and expression with COPD. Therefore, MMP7 responsible for degradation of elastin has been strongly linked to the progression of COPD.

An efficient evaluation system for factors affecting the genome editing efficiency in mouse.

Genome editing technology is widely used in the field of laboratory animal science for the production of genetic disease models and the analysis of gene function. One of the major technical problems in genome editing is the low efficiency of precise knock-in by homologous recombination compared to simple knockout via non-homologous end joining. Many studies have focused on this issue, and various solutions have been proposed; however, they have yet to be fully resolved. In this study, we established a system that can easily determine the genotype at the mouse (Mus musculus) Tyr gene locus for genome editing both in vitro and in vivo. In this genome editing system, by designing the Cas9 cleavage site and donor template, wild-type, knockout, and knock-in genotypes can be distinguished by restriction fragment length polymorphisms of PCR products. Moreover, the introduction of the H420R mutation in tyrosinase allows the determination of knock-in mice with specific coat color patterns. Using this system, we evaluated the effects of small-molecule compounds on the efficiency of genome editing in mouse embryos. Consequently, we successfully identified a small-molecule compound that improves knock-in efficiency in genome editing in mouse embryos. Thus, this genome editing system is suitable for screening compounds that can improve knock-in efficiency.

Modification of a previously patented method to unequivocally score A2-like and A1-like bovine ß-casein variants.

A growing interest in the production and commercialization of A2 cow's milk has been observed in many countries in the last few years due to the beneficial properties for human health attributed to A2 ß-casein variant. Methods of varying complexity and different equipment requirements have been proposed for the determination of the ß-casein genotype of individual cows. We proposed herein a modification of a previously patented method based on an amplification-created restriction site PCR followed by restriction fragment length polymorphism analysis. This method allows to identify and differentiate A2-like from A1-like ß-casein variants, after differential endonuclease cleavage flanking the nucleotide that determines the amino acid at position 67 of ß-casein. The advantages of this method are that it: • enables to unequivocally score A2-like as well as A1-like ß-casein variants, • can be performed at low cost in simply equipped molecular biology laboratories, and • can be scaled up to analyze hundreds of samples per day. For these reasons, and based on the results obtained from the analysis carried out in this work, it showed to be a reliable method for the screening of herds to selective breeding of homozygous cows and bulls for A2 or A2-like alleles.

Association of single-nucleotide polymorphisms in dual specificity phosphatase 8 and insulin-like growth factor 2 genes with inosine-5'-monophosphate, inosine, and hypoxanthine contents in chickens.

OBJECTIVE: This study aimed to identify the single-nucleotide polymorphisms (SNPs) in the dual-specificity phosphatase 8 (DUSP8) and insulin-like growth factor 2 (IGF2) genes and to explore their effects on inosine-5'-monophosphate (IMP), inosine, and hypoxanthine contents in Korean native chicken -red-brown line (KNC-R Line). METHODS: A total sample of 284 (males, n = 127; females n = 157) and 230 (males, n = 106; females, n = 124) aged of 10 weeks old KNC-R line was used for genotyping of DUSP8 and IGF2 genes, respectively. One SNP (rs313443014 C>T) in DUSP8 gene and two SNPs (rs315806609A/G and rs313810945T/C) in IGF2 gene were used for genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and KASP methods, respectively. The Two-way analysis of variance of the R program was used to associate DUSP8 and IGF2 genotypes with nucleotide contents in KNC-R chickens. RESULTS: The DUSP8 (rs313443014 C>T) was polymorphic in KNC-R line and showed three genotypes: CC, CT, and TT. The IGF2 gene (rs315806609A/G and rs313810945T/C) was also polymorphic and had three genotypes per SNP, including GG, AG, and AA for the SNP rs315806609A/G and genotypes: CC, CT, and TT for the SNP rs313810945T/C. Association resulted into a strong significant association (p<0.01) with IMP, inosine, and hypoxanthine. Moreover, the significant effect of sex (p<0.05) on nucleotide content was also observed. CONCLUSION: The SNPs in the DUSP8 and IGF2 genes might be used as genetic markers in the selection and production of chickens with highly flavored meat.

Association of IL-17A promoter region SNP-rs2275913 with urinary bladder cancer.

Objective: Urinary bladder cancer (UBC) is the fourth most common cancer among men and tenth most common cancer in women. This study investigated an association of interleukins -17A promoter region single nucleotide polymorphism (SNP)-rs2275913 with UBC in Pakistani population. Methods: Population-based study was designed with 127 UBC patients and 100 healthy individuals. Only UBC Patients were included and other diseases hepatitis or any other malignancy/cancer were excluded from the study. Polymerase chain reaction Restriction fragment length polymorphism technique was used to genotype the rs2275913 SNP in patients and control. Linear regression analysis was performed on the genotype data and allelic frequency data. Online statistical tool was used to calculate ratio of odds. Results: Linear regression analysis showed that there was no association between rs2275913 SNP and UBC patients in the dominant model (OR = 0.815, CI = 0.415-1.6), recessive model (OR = 0.389, CI = 0.014-5.565), codominant model (OR = 0.376, CI=0.013-5.420) and (OR = 0.855, CI = 0.427-1.713). Moreover, among the UBC samples, low-grade non-muscle invasive UBC samples dominant model (OR = 0.722, CI = 0.316-1.637), recessive model (OR = 0.000, CI = 0.000-5.864), codominant model (OR = 0.864, CI = 0.030-12.668), and (OR = 0.788, CI = 0.341-1.806) did also not show any association. When same analysis was performed for high-grade muscle invasive UBC, dominant (OR = 0.936, CI = 0.403-2.155), recessive model (OR = 0.875, CI = 0.031-12.696), and codominant model (OR = 0.864, CI = 0.030-12.668,), and (OR = 0.942, CI = 0.394-2.232) did not show any association. Conclusion: Results revealed that rs2275913 did not show any associated with the high risk of UBC in Pakistani population. Some limitations of the studies are firstly, the samples size and other are detailed information on UBC and role of inflammation.

A Novel 16S rRNA PCR-Restriction Fragment Length Polymorphism Assay to Accurately Distinguish Zoonotic Capnocytophaga canimorsus and C. cynodegmi.

The zoonotic bacteria Capnocytophaga canimorsus and C. cynodegmi, the predominant Capnocytophaga species in the canine oral biota, can cause human local wound infections or lethal sepsis, usually transmitted through dog bites. Molecular surveying of these Capnocytophaga species using conventional 16S rRNA-based PCR is not always accurate due to their high genetic homogeneity. In this study, we isolated Capnocytophaga spp. from the canine oral cavity and identified them using 16S rRNA and phylogenetic analysis. A novel 16S rRNA PCR-restriction fragment length polymorphism (RFLP) method was designed based on our isolates and validated using published C. canimorsus and C. cynodegmi 16S rRNA sequences. The results showed that 51% of dogs carried Capnocytophaga spp. Among these, C. cynodegmi (47/98, 48%) was the predominant isolated species along with one strain of C. canimorsus (1/98, 1%). Alignment analysis of 16S rRNA sequences revealed specific site nucleotide diversity in 23% (11/47) of the C. cynodegmi isolates, which were misidentified as C. canimorsus using previously reported species-specific PCR. Four RFLP types could be classified from all the isolated Capnocytophaga strains. The proposed method demonstrates superior resolution in distinguishing C. cynodegmi (with site-specific polymorphism) from C. canimorsus and especially in distinguishing C. canimorsus from other Capnocytophaga species. After in silico validation, this method was revealed to have an overall detection accuracy of 84%; notably, accuracy reached 100% in C. canimorsus strains isolated from human patients. Overall, the proposed method is a useful molecular tool for the epidemiological study of Capnocytophaga in small animals and for the rapid diagnosis of human C. canimorsus infections. IMPORTANCE With the increased number of small animal breeding populations, zoonotic infections associated with small animals need to be taken more seriously. Capnocytophaga canimorsus and C. cynodegmi are part of common biota in the mouths of small animals and can cause human infections through bites or scratches. In this study, C. cynodegmi with site-specific 16S rRNA sequence polymorphisms was erroneously identified as C. canimorsus during the investigation of canine Capnocytophaga by conventional PCR. Consequently, the prevalence of C. canimorsus is incorrectly overestimated in epidemiological studies in small animals. We designed a new 16S rRNA PCR-RFLP method to accurately distinguish zoonotic C. canimorsus from C. cynodegmi. After validation against published Capnocytophaga strains, this novel molecular method had high accuracy and could detect 100% of C. canimorsus-strain infections in humans. This novel method can be used for epidemiological studies and the diagnosis of human Capnocytophaga infection following exposure to small animals.

Characterization of polymorphisms in the follicle-stimulating hormone receptor and insulin-like growth factor-1 genes and their association with fertility traits in Jawa-Brebes cows.

Background and Aim: The availability of fertility markers is crucial for maintaining, protecting, and improving the genetics of Jawa-Brebes (Jabres) cows. Follicle-stimulating hormone receptor (FSHR) and insulin-like growth factor-1 (IGF-1) play critical roles in female reproductive physiology. The single-nucleotide polymorphisms (SNPs) FSHR G-278A and IGF-1 C-512T correlate with cows' fertility traits. This study aimed to identify these SNPs and their potential associations with fertility parameters in Jabres cows. Materials and Methods: Samples were collected from 45 heads of multiparous Jabres cows aged 3-10 years with body condition scores of 2.5-5.0 on a 5-point scale in Brebes Regency, Java, Indonesia. These cows were assigned to fertile (n = 16) and infertile groups (n = 29). Polymerase chain reaction (PCR) was carried out for DNA amplification of FSHR G-278A and IGF-1 C-512T fragments. Restriction fragment length polymorphism-PCR with the restriction enzymes FaqI for the product of FSHR G-278A and SnaBI for the product of IGF-1 C-512T was used to identify SNPs. Results: The FaqI enzyme cut the 211 bp DNA fragment of FSHR G-278A in all samples into two bands of 128 bp and 83 bp (GG genotype). Meanwhile, the genotyping of amplicon products of IGF-1 C-512T generated a single 249 bp fragment (CC genotype) in both groups. Conclusion: The results showed that the FSHR G-278A/FaqI and IGF-1 C-512T/SnaBI loci were monomorphic in Jabres cows. Thus, neither FSHR G-278A/FaqI nor IGF-1 C-512T/SnaBI is a possible genetic marker for fertility in Jabres cows.

To Study the ß-Globin Haplotype Pattern of Descent of a Set of Linked Alleles Occurring on the Same Chromosome in the Northern Province of India.

OBJECTIVE: To study the five mutations commonly prevalent in North India, i.e., IVS-I-5 (G→C), 619 bp deletion, IVS-I-1 (G→T), codon 41/42 (-TTCT), and codon 8/9 (+G), in the beta thalassemia (ß-thalassemia) major children. The specific ß-thalassemia mutations of different haplotype patterns of the ß-globin gene cluster will also be determined. METHODS: A total of 125 children diagnosed with ß-thalassemia major visiting the Department of Pediatrics of King George's Medical University were involved in the study. As per the QIAamp (Qiagen, Hilden, Germany) manufacturer guidelines, genomic DNA was isolated from whole blood. To identify the haplotype pattern within the ß-globin gene cluster, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used. The respective restriction endonucleases used were Hind III/GƔ, Hinc II/Ψß, Hinf I/ß, Ava II/ß, and BamHI for the haplotype analysis in the ß-globin pattern of descent of a set of linked alleles occurring on the same chromosome. RESULTS: Among the five common mutations, 73 patients had IVS-I-5 (G→C), 28 patients had 619 bp deletion, 17 patients had IVS-I-1 (G→T), five patients had Cd 41/42 (-TTCT), and two patients had Cd 8/9 (+G) mutations. Fifteen haplotypes (haplotypes 1-15) were identified in 125 ß-thalassemia major children. Among the five haplotypes observed in the IVS-I-5 (G→C) mutation, the H1 haplotype was most predominant with a frequency of 27.2%, followed by the H2, H4, H3, and H10 haplotypes in the given population. In 619 bp deletion, IVS-I-1 (G→T), codon 41/42, and codon 8/9, haplotype H9, H12, H11, and H5 were seen, respectively. CONCLUSION: ß-thalassemia was found to be the most common in the northern province of Uttar Pradesh. The linkage of ß-globin gene haplotypes with ß-thalassemia mutations was explored in the northern province of Uttar Pradesh. The population of different natives is being mixed up due to migration and industrialization. These were some reasons for the occurrence of haplotypic heterogeneity. This haplotype heterogeneity was correlated with the origin of these mutations found to be unlike the origin of common ones from different provinces.