Relationship Between IL-10 Single Nucleotide Polymorphisms (rs1800871, rs1800872, and rs1800896) and the Severity of COVID-19.

Background: Interleukin-10 (IL-10) is an anti-inflammatory cytokine whose levels are elevated in patients with severe COVID-19. IL-10 polymorphisms may play a role in increasing IL-10 levels and the severity of COVID-19. This study aimed to investigate the relationship between IL-10 single nucleotide polymorphisms (SNPs) (rs1800896 [-1082 C < T], rs1800871 [-819 A > G], and rs1800872 [-592 T > G]) and the severity of COVID-19 in patients from Kermanshah Province, Iran. Methods: A total of 150 patients with mild COVID-19 (84 men and 66 women aged 40.1 ± 12.44 years) and 143 patients with severe COVID-19 (76 men and 67 women aged 61.04 ± 15.65 years) participated in this study. Blood samples were collected from the patients, DNA was extracted, and the genotype of each SNPs was determined using the polymerase chain reaction-restriction fragment length polymorphism method. Result: The results of this study did not show a significant relationship between the genotypes of the three studied SNPs and the severity of COVID-19 (p > 0.05). Conclusion: According to our findings, these SNPs were not associated with COVID-19 severity in patients in Kermanshah.

Nitric Oxide Synthase 3 Gene Polymorphisms and Their Association with Acute Myocardial Infarction and Chronic Stable Angina: A Case-Control Study from Northern India.

Background: Coronary artery disease (CAD) that encompasses acute myocardial infarction (AMI), chronic stable angina (CSA), and unstable angina (UA) has numerous known risk factors. Genetic predispositions contribute as major risk in the development of CAD and the genes regulating atherosclerosis are important for disease prevention. Nitric oxide synthase 3 (NOS3) gene responsible for nitric oxide (NO) production is of special importance. Aim: To evaluate the role of three NOS3 polymorphisms (-786C/T, 894G/T, and 4a4b) in patients with CAD, particularly in AMI and CSA and their comparison with healthy controls. Materials and Methods: One hundred patients in each AMI and CSA group and 100 controls were included and were typed for three NOS3 polymorphisms (-786C/T, 894G/T, and 4a4b) by polymerase chain reaction-restriction fragment length polymorphism. Plasma NO metabolites (NOx) were also evaluated. Results: A significant association of 894G/T polymorphism with AMI in dominant model (P = 0.052) and with CSA in dominant and codominant models was detected (P = 0.008 and P = 0.006, respectively). Plasma NO levels were found to be significantly higher (P < 0.0001) in healthy controls (43.80 ± 6.28) compared to AMI and CSA patients (37.05 ± 6.75 and 38.67 ± 5.61). No significant association of -786C/T and 4a4b polymorphism with AMI and CSA risk under recessive, dominant, and codominant models was detected. Conclusion: Our study revealed a significant association of 894G/T polymorphism with AMI and independent association of NOx levels with CAD, indicating high risk of CAD in the North Indian population. Our findings will be helpful in identifying the genetic risk factors associated with CAD and better management of the diagnostic as well as therapeutic measures.

[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.

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.

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.

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.

The Taq 1 polymorphism of Vitamin D receptor gene is associated with oral cancer and preoral cancer in North Indian population.

Background: Oral cancer is known as one of the most common cancers, with a poor prognosis, related to delayed clinical diagnosis, either due to the lack of particular biomarkers related to the disease or costly therapeutic alternatives. Aims and Objectives: In this study association of single nucleotide polymorphism (Taq1, T>C) in Vitamin D receptor gene with oral cancer and pre oral cancer was studied. Materials and Methods: Total 230 patients of precancerous oral lesions (Leukoplakia 70, Oral Sub mucous fibrosis 90, Lichen Planus 70), 72 oral cancer patients and 300 healthy control subjects were genotyped by PCR-RFLP methods. Chi-square test was used for calculation of genotype and allele frequencies. Results: Mutant genotype CC as well as C allele were found to significantly decrease the risk of oral disease (P value=0.04, OR=0.60 and P value=0.02, OR=0.75 respectively). In particular, compared to non smokers, smokers with TC & CC genotypes were at decrease risk of oral diseases (P value=0.0001, OR=0.04). The mutant allele genotype CC as well as the mutant allele C showed protective association with leukoplakia (P value=0.01, OR=0.39 & P value=0.009, OR=0.59 respectively). However, individual with CC genotype had developed high cell differentiated grade at diagnosis (OR= 3.78, P value= 0.008). Conclusions: This study concludes that VDR (Taq1) polymorphism is associated with oral cancer and pre oral cancer susceptibility in North Indian population.

Genetic polymorphism of prolactin and nitric oxide synthase in Holstein cattle.

Background and Aim: Bacterial and viral infections affect the welfare of animals and lead to large economic losses in dairy cattle breeding due to decreased productive indicators and increased culling rates. In modern dairy farming, farmers are looking for effective solutions to prevent and minimize infectious disease risks. To this end, the most relevant study field is the search for gene sites that impact production and health. This study aimed to determine the nature of the distribution of the relative frequencies of alleles and genotypes of polymorphic prolactin (PRL) and nitric oxide synthase (NOS2) in Holstein cows and identify the relationship of these genes with resistance to mastitis and bovine leukemia. Materials and Methods: For this study, we chose cows because infectious diseases affect the amount of lactation and milk quality. Holstein cattle with mastitis and bovine leukemia were selected. Animal genotypes were determined by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. The results were analyzed using a nonparametric statistical method using Microsoft Excel 2010 and Statistica 6.0. Results: In healthy animals, 94 genotypes were identified for both genes under study. For bPRL, bPRL-RsaIAA (72) was the most common genotype and bPRL-RsaIBB (4) the least; for NOS2, bNOS2 -HinfIAB (47) was the most common genotype and bNOS2 -HinfIAA the least (21). In animals with leukemia, 34 genotypes were identified. For PRL, bPRL-RsaIAA (25) was the most common genotype and bPRL-RsaIBB (2) the least; for NOS2, bNOS2 -HinfIBB (17) was the most common genotype and bNOS2 -HinfIAA (3) the least. In animals with mastitis, 67 genotypes were identified. For PRL, bPRL-RsaIAA (43) was the most common genotype and bPRL-RsaIBB (6) the least; for NOS2, bNOS2 -HinfIBB (31) was the most common genotype and bNOS2-HinfIAA (7) the least. The distribution of genotypes of polymorphic bPRL and bNOS2 generally coincides, and bPRL-RsaIBB is the most common genotype. In groups of sick animals, the number of bNOS2 -HinfIAA homozygotes was lower than that of the control group. In particular, the proportion of animals with the bNOS2 -HinfIAA genotype with bovine leukemia was 8.7% and with mastitis was 10.3% compared with 22.4% in healthy animals. These data support the possible association of the bNOS2 -HinfIAA genotype with resistance to infection. The frequency of the bPRL-RsaIB allele was higher in groups of sick animals. This allele is associated with increased milk productivity, suggesting that highly productive animals are less resistant to the incidence of viral bovine leukemia and mastitis of bacterial etiology. Conclusion: DNA amplification of Holstein cattle for the polymorphic regions of PRL and NOS2 using the PCR-RFLP method revealed a possible connection between the distribution of relative allele frequencies of bPRL and bNOS2 and resistance to viral and bacterial infections. Thus, in groups of sick animals, the frequency of bPRL-RsaIBB, associated with increased milk production compared with the theoretically calculated equilibrium value was higher and the number of homozygotes bNOS2 -HinfIAA was lower than in the control group. In conclusion, animals with increased milk production were more prone to diseases, such as mastitis and bovine leukemia.

Eight single nucleotide polymorphisms and their association with food habit domestication traits and growth traits in largemouth bass fry (Micropterus salmoides) based on PCR-RFLP method.

Background: The largemouth bass (Micropterus salmoides), an economically important freshwater fish species widely farmed in China, is traditionally cultured using a diet of forage fish. However, given the global decline in forage fish fisheries and increasing rates of waterbody pollution and disease outbreaks during traditional culturing, there is a growing trend of replacing forage fish with formulated feed in the largemouth bass breeding industry. The specific molecular mechanisms associated with such dietary transition in this fish are, nevertheless, poorly understood. Methods: To identify single nucleotide polymorphisms (SNPs) related to food habit domestication traits and growth traits in largemouth bass fry, we initially genotyped fry using eight candidate SNPs based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, with genetic parameters being determined using Popgen32 and Cervus 3.0. Subsequently, we assessed the associations between food habit domestication traits of largemouth bass fry and these SNPs using the Chi-square test or Fisher's exact test. Furthermore, we used a general linear model to assess the relationships between the growth traits of largemouth bass fry and these SNPs. The Pearson correlation coefficient between growth traits and the SNPs was also determined using bivariate correlation analysis in IBM SPSS Statistics 22. Finally, the phenotypic variation explained (PVE) by the SNPs was calculated by regression analysis in Microsoft Excel. Results: The genotyping results obtained based on PCR-RFLP analysis were consistent with those of direct sequencing. Five SNPs (SNP01, SNP02, SNP04, SNP05, and SNP06) were found to be significantly correlated with the food habit domestication traits of fry (P < 0.05); SNP01 (P = 0.0011) and SNP04 (P = 0.0055) particularly, had showed highly significant associations. With respect to growth traits, we detected significant correlations with the two SNPs (SNP01 and SNP07) (P < 0.05), with SNP01 being significantly correlated with body length, and height (P < 0.05), and SNP07 being significantly correlated with body height only (P < 0.05). Conclusions: Our findings indicated that the PCR-RFLP can be used as a low-cost genotyping method to identify SNPs related to food habit domestication and growth traits in largemouth bass, and that these trait-related SNPs might provide a molecular basis for the future breeding of new varieties of largemouth bass.

Genotypic analysis of the FAS2-F1279Y (3836T>A) polymorphism conferring high ethyl caprylate productivity in industrial sake yeast strains.

In Saccharomyces cerevisiae, ethyl caprylate is produced by the esterification of caprylic acid, which is synthesized through the action of fatty acid synthase. A recent study reported a yeast mutant with a single nucleotide substitution in the alpha subunit of fatty acid synthase (FAS2) gene (F1279Y; 3836T>A) that produced large amounts of ethyl caprylate. Here, we designed two primer sets (P1/P2 and P3/P4) with mismatches that incorporate restriction sites for the enzymes NdeI and SspI, respectively and developed an easy and rapid polymerase chain reaction-restriction fragment length polymorphism assay to identify yeasts harboring the FAS2-F1279Y mutation associated with high ethyl caprylate productivity.

Identification of Arisaematis Rhizoma and Pinelliae Rhizoma by PCR-RFLP / 中国实验方剂学杂志

ObjectiveIn recent years， with the sharp decline of wild resources in Arisaematis Rhizoma and Pinelliae Rhizoma and the immaturity of medicinal cultivation technology， their adulterants have appeared frequently in the market， and the main identifying characteristics have mostly disappeared in the circulation of medicinal materials. Therefore， there is an urgent need to establish a molecular identification method that can quickly and effectively identify the specificity of Arisaematis Rhizoma and Pinelliae Rhizoma. MethodAfter comparison of the rbcL sequences of Arisaematis Rhizoma，Pinelliae Rhizoma， and their adulterants， the specific enzyme cleavage sites Hae Ⅲ and Dra Ⅰ of Arisaematis Rhizoma and Pinelliae Rhizoma， respectively， were selected and identified by polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）. The main system conditions of PCR-RFLP reaction were established and optimized， and their durability and the ability to detect genuine， adulterants， and mixed counterfeits were investigated. ResultThe PCR-RFLP identification method of Arisaematis Rhizoma and Pinelliae Rhizoma was established. After specific primer amplification， Arisaematis Rhizoma and Pinelliae Rhizoma could be digested by Hae Ⅲ and Dra Ⅰ-restricted endonucleases respectively， at annealing temperature of 54 ℃， the number of cycles of 35， and the amount of DNA template of 3-30 ng， producing two fragments or small cut fragments with a single band between 100-250 bp， whereas the mixed counterfeits were not cleaved and both showed a band at 250 bp. The method is highly accurate in identifying adulterants and mixed counterfeits of Arisaematis Rhizoma or Pinelliae Rhizoma. ConclusionThe PCR-RFLP method developed in this study allows for the rapid identification of Arisaematis Rhizoma and Pinelliae Rhizoma.

Relationship between Fok polymorphism of vitamin-D receptor gene and hypertensive disorder complicating pregnancy in Han nationality of Qinghai province / 解剖学报

[ Abstract] Objective To investigate the relationship between single nucleotide polymorphism (SNP) Fok (rs2228570 / rs10735810) of vitamin D receptor (VDR) gene and hypertensive disorder complicating pregnancy (HDCP) in Han nationality women of Qinghai province. Methods A total of 137 Han nationality HDCP subjects (HDCP group) and 146 Han nationality normal pregnant subjects (control group) were selected from Qinghai province. The Fok polymorphism typing in HCDP group and control group was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) . The mutation was confirmed by sequencing. SPSS 19. 0 statistical software was used to test whether there were significant differences between two groups in general clinical data, genotype and allele frequency distribution. Results The frequency of FF Ff ff genotype of Fok in HDCP group and control group were 51. 82%, 37. 96%, 10. 22% and 34. 93%, 43. 15%, 21. 92% respectively (

Molecular analysis of interleukin 10 promoter region-592A/C polymorphism in hypertensive disorders of pregnancy in Qinghai Han nationality / 解剖学报

Objective To investigate the association between the interleukin 10 (IL-10) gene promoter region-592A/C (rs1800872) polymorphism and hypertensive disorders of pregnancy (HDP) in Han women of Qinghai province and to determine the expression of this gene in two groups (HDP group and healthy control group) preliminarily. Methods A total of 140 HDP patients (HDP group) and 140 normal pregnant women (control group) in Qinghai Province were selected. Using blood DNA as template, the IL-10-592A/C polymorphism typing of HDP group and control group was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and verified by sequencing. The expression of IL-10 mRNA in the placental tissues of the two groups was detected by Real-time PCR. Plasma IL-10 levels of the two groups were detected by ELISA. Results The frequencies of AA, AC and CC genotypes of IL-10 gene in HDP group and control group were 24. 29%, 44. 29%, 31. 42% and 13. 57%, 41. 43%, 45. 00% respectively, the difference in genotype distribution between the two groups was statistically significant (P<0. 05);AA genotype frequency in HDP group(24. 29%)was higher than that of control group(13. 57%)(P<0. 05), CC genotype frequency in HDP group (31. 42%) was lower than that in control group (45. 00%) (P < 0. 05), while there was no significant difference in genotype frequency of AC between the two groups (P<0. 05); The distribution of A and C allele frequencies of IL-10592A/C polymorphism was different between the two groups, and the A allele frequency of HDP group was higher than that of control group (

Endometrial receptivity defects MUC-1 and COX-2 polymorphisms in endometriosis.

The endometrium produces MUCIN-1 (MUC-1) and cyclooxygenase-2 (COX-2), which are essential for implantation. MUC-1 is required for adhesion, while COX-2 is necessary for decidualization. Variations or polymorphisms in MUC-1 and COX-2 can lead to changes in endometrial receptivity. This study investigated the relationship between MUC-1 and COX-2 polymorphisms and endometrial receptivity in endometriosis patients. Blood DNA samples were collected from 35 patients with endometriosis and 32 healthy patients between days 19 to 24 of their menstrual cycle (secretory phase). MUC-1 polymorphism was determined using the Amplification Refractory Mutation System (ARMS), and COX-2 gene polymorphism was assessed using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The frequency distribution of gene polymorphisms between the two groups was compared using bivariate analysis. There were seven genotypic combinations of MUC-1 and COX-2: AAGC; AAGG; GACC; GAGC; GAGG; GGGC; GGGG. The AAGC genotype combination test was significant, with an OR=6.43 (95% CI:1.09-7.62) and p=0.01. In conclusion, combining MUC-1 and COX-2 (AAGC) genotypes results in endometrial receptivity defects in endometriosis.

Comparison of culture and polymerase chain reaction-restriction fragment length polymorphism for identification of various Capnocytophaga species from subgingival plaque samples of healthy and periodontally diseased individuals.

Introduction: Capnocytophaga are facultative anaerobic Gram-negative bacilli and recognized as opportunistic pathogens of various extraoral infections. Only a few studies attempted to identify all the seven species of Capnocytophaga phenotypically and genotypically in healthy individuals and patients with chronic periodontitis. Studies to determine the prevalence of Capnocytophaga in subgingival plaque samples from healthy individuals, chronic gingivitis and periodontitis among Indian population are lacking. Aim: The aim of this study was to identify and compare the presence of Capnocytophaga species phenotypically through microbial culture and biochemical tests and genotypically through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in subgingival plaque of healthy individuals and patients with chronic gingivitis and chronic periodontitis. Materials and Methods: A total of 300 subjects, 100 each with gingivitis, periodontitis and periodontally healthy gingiva subjected, were included. Subgingival plaque was collected and was cultured for phenotypic identification (microbial culture and biochemical test), and for genotypic identification, DNA extraction was done and PCR-RFLP analysis was performed to identify the genus Capnocytophaga and also to identify different species of Capnocytophaga. Results: Of 300 individuals, Capnocytophaga species were identified from 237 (79%) individuals by PCR and 82 (27.33%) by culture. The prevalence of Capnocytophaga ochracea was found to be higher with both the methods followed by Capnocytophaga gingivalis and Capnocytophaga granulosa. Capnocytophaga genospecies, Capnocytophaga leadbetteri and Capnocytophaga Sputigena were isolated only by culture with very low prevalence that is 1.33%, 1.33% and 0.66%, respectively. We could not get any isolate of Capnocytophaga haemolytica by any of the two methods. Conclusion: Capnocytophaga species could be found in gingival sulci as well as periodontal pockets and can be detected by culture and PCR-RFLP. However, higher prevalence of these species in healthy compared to disease requires further analysis to determine their role in healthy and diseased periodontium.

Comparison of gdh polymerase chain reaction-restriction fragment length polymorphism and tpi assemblage-specific primers for characterization of Giardia intestinalis in children.

Background: Giardia is a diarrheagenic eukaryotic parasite that consists of at least eight morphologically identical but genetically distinct genotypes. Human giardiasis is caused mainly by A and B assemblages. Aim and objectives: The study aimed to compare the performance of gdh polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tpi assemblage-specific primers in genotyping of G. intestinalis. Materials and Methods: Stool samples of 315 children were microscopically screened for G. intestinalis. Positive samples were genotyped using tpi assemblage-specific primers and gdh semi-nested PCR-RFLP techniques. Results: The prevalence of Giardia was 18.1%. The detected genotypes using tpi and gdh approaches were assemblage A (15.8% vs. 12.7%) and assemblage B (36.8% vs. 74.5%) as single infections and mixed assemblages A and B (47.4% vs. 12.7%). The two approaches showed a moderate agreement (kappa index = 0.413, P < 0.001). PCR-RFLP of gdh gene revealed that sub-assemblages BIII and BIV were equally detected (30.9% each). The remaining samples were equally divided between sub-assemblage AII, mixed BIII and BIV, and mixed AII and BIII (12.7% each). A significant association was detected between the retrieved sub-assemblages and the presence of symptoms. Conclusions: Although both approaches confirmed the predominance of assemblage B, the use of assemblage-specific primers is more effective in elucidating the true picture of mixed assemblage infection.

Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis.

Aims: Rapid and accurate identification of mycobacteria is important for the species-specific treatment of the disease. The aim of this study was the identification at the species level of 34 nontuberculous mycobacteria strains isolated from respiratory tract samples and 14 reference strains as by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Materials and Methods: Isolates derived from clinical specimens were subcultured in the Lowenstein-Jensen medium. Deoxyribonucleic acid isolation was carried out using the boiling method. PCR amplification was performed using primers specific to the hsp65 gene region. The PCR products were digested BstEII and HaEIII enzymes. All samples were studied comparatively by two different centers. Results: In our study, the most common species were found to be Mycobacterium intracellulare in 23.52% (8/34). The performance of the PCR-RFLP method in detecting mycobacteria was found to be 82.35%. Conclusions: The PCR-RFLP method is a rapid, cheap, and practical method for the identification of mycobacteria.