Sanger Sequencing Technique Research Articles

Calreticulin mutational analysis in patients with myelofibrosis

Objective Our study aimed to determine the occurrence of CALR and JAK-2 mutation in patients with myelofibrosis. Patients and methods This cross-sectional study was conducted in the Department of Hematology, Lady Reading Hospital, Peshawar, and Princess Nourah Bint AbdulRehman University, Saudi Arabia. Blood samples and data were collected from patients in the Hematology Department of Lady Reading Hospital, Peshawar, Pakistan. Nonprobability convenience sampling technique was used. All patients of JAK-2 V617F-positive and JAK-2 V617F-negative primary or secondary myelofibrosis due to polycythemia vera or essential thrombocythemia were included. After taking blood and bone marrow sampling, DNA extraction was done manually, and these samples were analyzed for CALR mutations by Sanger sequencing technique. Data were recorded and analyzed in SPSS, version 20. Results Of 118 patients of myelofibrosis, we have found two types of genetic variations. One is single nucleotide polymorphism (SNP) in the 3’ UTR variant and the other is a novel indel frameshift mutation in the form of p.Leu 367 Thr Fx 63. Among these 118 patients, 14.40% patients had the indel frameshift mutation, whereas 46.61% patients had SNPs. The remaining patients did not harbor any significant changes. Conclusion Our study concluded that most patients had a SNP in the 3’ UTR variant and a novel frameshift mutation. Further large-scale studies should be organized to determine the co-occurrence of calreticulin mutations in the JAK-2-positive patients of myelofibrosis. The influence of this coexistence on the phenotype and clinical course of myelofibrosis should also be studied to better understand the diagnosis and prognosis of the patients with myelofibrosis.

Recurrent KRAS, KIT and SF3B1 mutations in melanoma of the female genital tract

BackgroundMalignant melanoma of the female genital tract is relatively uncommon and accounts for 3–7% of all melanoma localizations. This study aimed to identify driver genes in melanoma of the female genital tract with the purpose of enhancing understanding of disease pathogenesis and identifying potential new therapeutic targets to develop effective therapies.MethodsKIT (CD117) and BRAF expression were detected immunohistochemically. Polymerase Chain Reaction (PCR) and Sanger sequencing techniques were performed to identify the mutational status of BRAF, NRAS, KRAS, NF1, KIT, PDGFRA and SF3B1 on 19 melanomas of the female genital tract, paired with 25 cutaneous melanomas, 18 acral melanomas and 11 melanomas of nasal cavity.ResultsSomatic variant analysis identified KRAS (6/19; 32%) as the most commonly mutated gene, followed by KIT (4/19; 21%), SF3B1 (3/19; 16%) and NRAS (1/19; 5%). None of the cases were found to harbor BRAF, NF1 and PDGFRA mutations in melanomas of the female genital tract. However, none of the cases were found to harbor SF3B1 and KIT mutations in cutaneous melanomas, acral melanomas and melanomas of nasal cavity. Recurrent KIT mutations, as well as mutations in the less frequently mutated genes NRAS and SF3B1, were exclusively detected in vulvovaginal melanomas, but not in tumors arising in the cervix. However, recurrent KRAS mutations were detected in similar frequencies in tumors of the vulva, vagina, and cervix. Additionally, recurrent KRAS and KIT mutations occurred predominantly in polygonal and epithelioid cell types of melanoma in the female genital tract. Immunohistochemistry revealed moderate or strong cytoplasmic CD117 expression in 6 of the 19 cases (31.6%).ConclusionsWe observed that gynecologic melanoma harbored distinct mutation rates in the KIT, BRAF, SF3B1, KRAS, and NRAS genes. Our findings support the notion that gynecologic melanoma is a distinct entity from non-gynecologic melanoma, and these findings offer insights into future therapeutic options for these patients.

Distinctive Molecular typing of 16S rRNA of Bacillus species isolated from farm settlement.

Distinctive Molecular typing of 16S rRNA of Bacillus species isolated from farm settlement.

Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines.

Gliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sanger sequencing assay to screen or TERTp mutations. We established the Sanger sequencing approach for the detection of TERTp mutations based on human glioma cell lines U251 and assessed the analytical validation by determining the accuracy, sensitivity, precision, and specificity. In our study, we verified the accuracy of Sanger sequencing by the real-time polymerase chain reaction method. Our data showed that TERTp mutations were detected at an analytical sensitivity of 10% per mutant. The precision and specificity validation also showed the desired results. In total, 147 glioma patients were investigated for TERTp mutations, and of each patient, clinical data and molecular characteristics were analyzed. We found that anaplastic oligodendroglioma had the highest frequency of TERTp mutations (66.7%). No differences in TERTp mutation frequency were observed between frozen tissue specimens and formalin-fixed and paraffin-embedded tissue. TERTp mutations were associated with older patients (≥45 years), whereas isocitrate dehydrogenase (IDH) mutations were inclined to a younger age (<45 years), frontal location, and pathologic stage II-III patients. IDH mutations were significantly associated with O6-methylguanine-DNA methyltransferase (MGMT) methylation (P = 0.003) and lower Ki-67 protein expression (P = 0.011). Moreover, MGMT methylation was enriched in IDH-mutant/TERTp-mutant gliomas, and Ki-67 protein expression was the highest in the IDH-wild type/TERTp-mutant group. Taken together, the findings of this study indicate the establishment of a rapid, precise, and practical Sanger sequencing technique for TERTp mutations in gliomas that may show promising results in clinical applications.

An Antisense Circular RNA Regulates Expression of RuBisCO Small Subunit Genes in Arabidopsis.

Circular RNA (circRNA) is a novel class of endogenous long non-coding RNA (lncRNA) and participates in diverse physiological process in plants. From the dataset obtained by high-throughput RNA sequencing, we identified a circRNA encoded by the sense strand of the exon regions spanning two RuBisCO small subunit genes, RBCS2B and RBCS3B, in Arabidopsis thaliana. We further applied the single specific primer-polymerase chain reaction (PCR) and Sanger sequencing techniques to verify this circRNA and named it ag-circRBCS (antisense and across genic-circular RNA RBCS). Using quantitative real-time PCR (qRT-PCR), we found that ag-circRBCS shares a similar rhythmic expression pattern with other RBCS genes. The expression level of ag-circRBCS is 10–40 times lower than the expression levels of RBCS genes in the photosynthetic organs in Arabidopsis, whereas the Arabidopsis root lacked ag-circRBCS expression. Furthermore, we used the delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) to deliver in vitro synthesized ag-circRBCS into Arabidopsis seedlings. Our results indicate that ag-circRBCS could significantly depress the expression of RBCS. Given that ag-circRBCS was expressed at low concentration in vivo, we suggest that ag-circRBCS may represent a fine-tuning mechanism to regulating the expression of RBCS genes and protein content in Arabidopsis.

ANTIBACTERIAL POTENCY OF THE COAT OF CITRUS SINENSIS (ORANGE) ON BACILLUS SPECIES

Background: In other to curb the resurgence of drug resistance, there is need to find cheaper, environmental friendly and readily available alternative chemotherapy for all patients for all ages. Extracts from plants has proven to be potent against wide range of pathogen such as bacteria, viruses, fungi and parasites of which Citrus sinensis is an important example. Both the aqueous and ethanoic extracts of Citrus sinensis were evaluated on some Bacillus species which were isolated from vegetables in nearby farm around the Oke Ogun Polytechnic Saki. Aim: The aim of this study was to evaluate the phytocompounds and antimicrobial activities of the coat of Citrus sinensis on some Bacillus species. Methods: The aqueous and ethanoic extract of the dried plant coat was evaluated for its antimicrobial activities on the isolated organisms using Kirby bauer disc diffusion method. The isolates were subjected to molecular identification using the polymerase chain reaction and subsequent Sanger sequencing techniques. There was high correlation between the isolates’ identity at all the levels and sequencing except in few. The FASTA format of the nucleotides sequences were submitted at the GenBank at NCBI database for accession numbers which were MW362290 (Bacillus cereus), MW362291 (Bacillus cereus), MW362292 (Bacillus weidmanii), MW362293 (Bacillus cereus), MW362294 (Bacillus cereus) and MW362295 (Bacillus thuringiensis). Results: The active phytocompounds present in the aqueous and ethanol solvent were saponin, alkaloid, flavonoid, tannin, coumarin, steroid, terpenoid, cardiac glycosides, quinones, anthraquinones and phenol while Anthocyanin was present only in aqueous extract. These extracts both showed high antibiotics activities on all the isolated organisms. All the extracts recorded considerable antibiotic potency against all the isolates making the extracts of Citrus sinensis coat a suspect containing active biocidal agents. Conclusion: The coat of Citrus sinensis showed high antimicrobial properties against some Bacillus species, the antimicrobial effects was as a result of active phytochemicals present in the coat. This study established biocidal activities in both aqueous and ethanolic extracts. Its utilization for its pharmaceutical would be of great advantage both economically and environmentally.

CORRECTION OF THE MUTATION CAUSE OF CYSTIC FIBROSIS (F508DEL) IN PULMONARY EPITHELIUS LINE CELLS BY CRISPR/CAS9

Cystic fibrosis is a genetic, autosomal recessive disease caused by mutations in the CFTR gene (cystic fibrosis transmembrane conductance regulator). The most common mutation is F508del with around 70% prevalence worldwide. Currently, the disease has no cure and the main symptoms are treated. The technique of the CRISPR/Cas has favorable prospects to correct the mutation in DNA and rescue function of CFTR protein, improving patient's quality of life. The main objective this work was to build molecular tools to correct the F508del mutation of cystic fibrosis in a pulmonary epithelial cell line (CFBE). The work is divided into stages: (1) Design of sgRNA and construction of tools and (2) edition evaluation by qPCR, PCR and Sanger sequencing techniques. The designs sgRNAs were selected based on the proximity of the mutation, number of off-targets and efficiency predicted. The insertion of the sgRNA sequence in the plasmid (px458) was confirmed by the PCR technique, digestion with restriction enzymes and sequencing. The transfection method selected was by lipid agents, showing an efficiency of about 9% in the entry of the plasmid into target cell. The built tool based ribonucleoprotein, had the RNA obtained by in vitro transcription. After formation of the complex with the purified protein (saCas9) this tool showed activity (30%) in recognizing and cleaving DNA in vitro. After 48 hours transfection cells, was made a sorting by flow cytometry and the correction confirmed by qPCR techniques, the detection the correction of the F508del mutation was below 12.5% or null, under the conditions tested. New assays will be performed included design modifications for this tool to obtain satisfactory data.

Application of DNA barcoding and metabarcoding for species identification in salmon products

ABSTRACT Mislabelling is a significant manifestation of food fraud. Traditional Sanger sequencing technology is the gold standard for seafood species identification. However, this method is not suitable for analysing processed samples that may contain more than one species. This study tested the feasibility of next-generation sequencing in identifying mixed salmon products. Salmon samples containing up to eight species were amplified using 16S rRNA mini-barcode primers, and sequenced on an Illumina HiSeq2500 platform. All species were accurately identified, and mixtures as low as 1% (w/w) could be detected. Furthermore, this study conducted a market survey of 32 products labelled as salmon. For pure and mixed fish products, Sanger and next-generation sequencing techniques were respectively used for species identification, and for NGS results, we also used real-time PCR method to cross-validate the mixed products to further verify the accuracy of the DNA metabarcoding technology established in this study. DNA barcoding and metabarcoding of commercial salmon food products revealed the presence of mislabelling in 16 of 32 (50%) samples. The developed DNA barcoding and metabarcoding methods are useful for the identification of salmon species in food and can be used for quality control of various types of salmon products.

Al-Gazali Skeletal Dysplasia Constitutes the Lethal End of ADAMTSL2-Related Disorders.

Lethal short-limb skeletal dysplasia Al-Gazali type (OMIM %601356), also called dysplastic cortical hyperostosis, Al-Gazali type, is an ultra-rare disorder previously reported in only three unrelated individuals. The genetic etiology for Al-Gazali skeletal dysplasia has up until now been unknown. Through international collaborative efforts involving seven clinical centers worldwide, a cohort of nine patients with clinical and radiographic features consistent with short-limb skeletal dysplasia Al-Gazali type was collected. The affected individuals presented with moderate intrauterine growth restriction, relative macrocephaly, hypertrichosis, large anterior fontanelle, short neck, short and stiff limbs with small hands and feet, severe brachydactyly, and generalized bone sclerosis with mild platyspondyly. Biallelic disease-causing variants in ADAMTSL2 were detected using massively parallel sequencing (MPS) and Sanger sequencing techniques. Six individuals were compound heterozygous and one individual was homozygous for pathogenic variants in ADAMTSL2. In one of the families, pathogenic variants were detected in parental samples only. Overall, this study sheds light on the genetic cause of Al-Gazali skeletal dysplasia and identifies it as a semi-lethal part of the spectrum of ADAMTSL2-related disorders. Furthermore, we highlight the importance of meticulous analysis of the pseudogene region of ADAMTSL2 where disease-causing variants might be located. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Increase in Mortality and Second Neoplasms in Chronic Lymphocytic Leukemia with Pro/Pro Genotype of TP53 Codon 72

Chronic Lymphocytic Lymphoma (CLL) is a heterogeneous disease in which many important factors for its prognosis have been identified. The normal functioning of p53 is one of the most critical barriers against cancer; therefore, if it has a deletion and/or mutation, it is a robust biomarker for the therapeutic response in CLL. The possibility was raised that some germline single - nucleotide polymorphisms of TP53 in healthy populations may also affect p53 function. One of the most studied polymorphisms of the TP53 gene is codon 72 in exon 4, a CGC to CCC transition (R72P), due to its potential effect on cancer risk. As with many types of cancer, its association with a worse prognosis in CLL is unclear. We analyzed the relationship of the genotypes of the TP53 codon 72 polymorphism in a large cohort of patients with CLL, to demonstrate the association of codon 72 with the evolution of the disease. Using the IDIPHIM patient database, 558 patients with a diagnosis of CLL were included, with clinical data, immunophenotype studies, FISH, IgHV, and karyotype, at the time of diagnosis and during follow-up. The TP53 codon 72 Arg/Arg, Arg/Pro, and Pro/Pro genotypes were analyzed using RT-PCR and Sanger sequencing techniques. After analyzing the sample of patients, 321 patients with the Arg/Arg genotype, 202 with the Arg/Pro genotype, and 35 with the Pro/Pro genotype were found. In the comparative analysis of the three groups, the patients with the Pro/Pro genotype had a higher number of patients in advanced stages B and C. The latter had a significant association with Binet staging (p = 0.002) compared to the other groups. Likewise, patients with the Pro/Pro genotype had a higher incidence of Richter transformation, whose association was significant (p = 0.013). Also, the patients who were within the Pro/Pro genotype group showed a significant association (p = 0.030) with the Time to the first treatment (TFT), also observing that the group of patients with the Arg/Pro genotype had a more considerable time until your first treatment. 19.7% (110/558) had a second neoplasm, having a significantly higher association with the homozygous groups (Arg/Arg and Pro/Pro) than with the Arg/Pro group, which on the contrary, had fewer second neoplasms (p = 0.016) (see Table 1). Regarding the type of tumors, we found 14.5% of the bladder, 14.5% of the skin, 14.5% of the colon, 13.6% of the prostate, and 12.7% of the lung. No associations were found between Codon 72 and CD38+, ZAP70+, complex karyotype, IgHV, NOTCH-1, del 11q, 12+, p53, del 13q, TP53 mutation. Still, when forming a group between the p53 deletion and TP53 mutation, if significant differences were found (p = 0.023), Pro / Pro group had the highest percentage. The overall survival was 156.32 months (139.92 - 172.72), showing that patients with the Arg/Pro genotype live 40 months more significantly than the other groups (p = 0.028) (see Figure 1). Finally, in the multivariate analysis, age, complex karyotype, 11q deletion, p53 deletion, unmutated IgHV, and Pro/Pro genotype at codon 72 were identified as independent variables associated with an increased risk of death (see Table 2). In conclusion, the Pro/Pro genotype of TP53 Codon 72 has a potential role in the progression and the higher mortality of patients with CLL. Conversely, the Arg / Pro genotype was associated with a lower incidence of second malignancies and higher overall survival. Disclosures No relevant conflicts of interest to declare.

Association of Exon 14 of the SOX6 Gene Sequence Variations with Response to Hydroxyurea Therapy in Patients Carrying Non Transfusion-Dependent Thalassemia

Hydroxyurea (HU) activates the γ-globin gene, resulting in increased Hb F synthesis. The SOX6 gene is a member of the Sox (Sry-type HMG box) family of transcription factors, characterized by minor groove binding domain. The DNA binding domain of this gene is encoded by exon 14. We assessed the relationship between response to HU and exon 14 of the SOX6 gene sequence variations in patients with non transfusion-dependent thalassemia (NTDT). One hundred NTDT patients from southern Iran underwent HU therapy randomly participated in this cross-sectional study between February 2013 and October 2014. Based on response to HU therapy, the patients were divided into two groups: good and poor responder. Sequence variations of exon 14 of the SOX6 gene was assayed by the Sanger sequencing technique. From all evaluated single nucleotide polymorphisms (SNPs) as above, we found no significant association between sequence variations of exon 14 of the SOX6 gene and response to HU therapy (p > 0.05). It seems that no SNPs in exon 14 of the SOX6 gene is associated with response to HU in NTDT patients, but more studies are needed for further evaluation.

Detection of a Larva of Armillifer armillatus in a Potto (Perodicticus potto) from the Republic of the Congo.

We determined the complete sequence of the mitochondrial DNA (mtDNA) of a parasite discovered between the subcutaneous tissue and the peritoneum of an African nocturnal non-human primate (NHP). The parasite and host sequences were obtained by a combination of Sanger sequencing and nanopore MinION techniques. Analyses of mtDNA gene arrangements and sequences unambiguously showed that the parasite investigated was the pentastomid Armillifer armillatus, also commonly named the tongue worm. The full-length mitochondrial genome of A. armillatus, measuring 16,706 bp in length, contains 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes, an arrangement identical to that of previously described pentastomid mitochondrial genomes. We describe here the second full mitochondrial genome of A. armillatus to date. To identify the NHP host, maximum likelihood phylogenetic analyses of a 441-bp fragment on the 12S rDNA gene and of a 1,140-bp fragment of the mitochondrial cytochrome b strongly support clustering with the African lorisid Perodicticus potto, a species that has rarely been reported as an intermediate host of this parasite.

Identification and characterization of SNPs in released, landrace and wild accessions of mungbean (Vigna radiata (L.) Wilczek) using whole genome re-sequencing

Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] is vital grain legume having nutritional and socio-economic importance, especially in the developing countries. We performed whole-genome re-sequencing of three accessions representing the wild progenitor species, released and landrace of mungbean to identify SNPs with relevance to genetic relationships analysis. Approximately 9.3 million raw reads were obtained using Ion Torrent PGM™ platform and more than 92% of the reads were mapped to the reference mungbean genome. We identified a total of 233,799 single-nucleotide polymorphisms in relation to the reference genome (SNPs: 103,341 in wild, 93,078 in released and 37,380 in landrace accessions) and 9544 insertions and deletions (InDels: 4742 in wild, 3608 in released and 1194 in landrace accessions) in the coding and non-coding regions. In all accessions, genomic variants were unevenly distributed within and across the mungbean chromosomes. Among these 5339, 4739 and 1795 SNPs were non-synonymous in 815, 790 and 317 genes of wild, released and landrace accessions, respectively. These polymorphisms might contribute to the variation in important pathways of genes for abiotic and biotic stress tolerance and important agronomic traits, such as seed dormancy, flowering time and seed size in mungbean. Among the randomly selected SNPs, a selected subset was validated using Sanger sequencing technique. The genomic variations among mungbean wild, released and landrace accessions constitute a powerful tool to support genetic research and molecular breeding of mungbean.

Molecular characterization of viruses causing maize lethal necrosis disease in South-Rift region, Kenya

The first report of Maize lethal necrosis (MLN) disease in Africa was in Bomet, Kenya, nine years ago. It has since spread in East and Central African (ECA) countries, causing massive yield losses. Currently, 90% of the preferred commercial maize germplasm grown by farmers in Kenya is susceptible to MLN disease. As such, the disease has continued to pose a serious challenge to food security in the ECA region. This study sought to characterize the MLN, causing viruses present in the maize leaf samples collected from the South-Rift region. Using total RNA extracted from 60 leaf samples collected from Bomet, Kericho, and Kisumu Counties, reverse transcription-polymerase chain reaction (RT-PCR) was carried out. The PCR products with the strongest bands were purified and sequenced using the Sanger sequencing technique. The results showed that samples from the three counties were positive for maize chlorotic mottle virus (MCMV) (MH645622 and MH645621) and sugarcane mosaic virus (SCMV) (MH645623, MH645624 and MH645625) and negative to wheat streak mosaic virus (WSMV). The coat protein (CP) sequences of MCMV isolates were closely related to the sequences of MCMV isolates, which had been previously reported from Eastern and Sub-Saharan Africa. For the CP sequences of SCMV isolates, only one sequence of the isolate KCO59 was similar to the sequence of a Kenyan isolate (JX286708). Sequences of isolates KCO5 and KCO24 were not identical to those of the Kenyan isolate (JX286708). Based on these results, in the surveyed counties, isolates of SCMV are genetically diverse, while those of MCMV are not. There exists a new variant of SCMV, which appears to be the main potyvirus in synergism with MCMV in causing MLN disease in Kenya. Key words: Maize lethal necrosis (MLN), maize chlorotic mottle virus (MCMV), sugarcane mosaic virus (SCMV), wheat streak mosaic virus (WSMV).

Antimicrobial resistance profile and antibacterial activity of ginger and garlic extract on diary isolated E. coli And Salmonella typhii

This study investigated the in vitro comparative assessment of antibacterial activity of ginger (Zingiber officinale) and garlic (Allium sativum). Four different extracts were prepared (ethanol, n-hexane, ethanol + waters, aqueous extracts) of ginger and garlic to determine the antibacterial action against rising spates of resistance to Escherichia coli and Salmonella spp. which were isolates from raw milk of cattle using disc diffusion method. The bacterial strains used was identified and characterized by standard bacteriological method (colonial morphology, biochemistry), PCR and Sanger Sequencing Techniques (molecular). Both garlic and ginger were identified at FRIN in Ibadan and then concentrated at Department of Pharmaceutical Chemistry, University of Ibadan. The extracts of both ginger and garlic showed remarkable resistance against both organisms with no inhibitory zone while the synthetic antibiotic used as control (Chloramphenicol) was sensitive against the two test microorganisms. The result obtained was subjected to statistical ANOVA at P≥ 0.05 level of significance. Although both plants have been reported to have anti-bacterial properties but this study contrasted this assertions making its recommendation as plants of choice in ameliorating bacterial infection as a cause for re-examination.

Terminal Genome Sequences of the Soft Tick Bunyavirus.

The complete genome sequence of the soft tick bunyavirus (STBV) was obtained using the Sanger sequencing technique. Comparison with other viral sequences revealed that STBV has unique sequences in the terminal regions that are highly conserved among the genus Orthonairovirus.

Activated PI3K-delta syndrome in an Egyptian pediatric cohort with primary immune deficiency

BackgroundActivated Phospho-Inositide 3 (PI3) Kinases Delta syndrome (APDS) can underlie primary immune deficiency. The prevalence and phenotypic characterization of these patients are not well described in Egypt. ObjectivesTo describe patients with APDS in hospitalized children with recurrent respiratory tract infections with suspected primary immune deficiency. Methods79 patients were included in the study. E1021K and E525K mutations of PI3K δ chain gene were screened by Sanger sequencing technique. Resultsone patient was heterozygous to E1021K mutation; a female child was diagnosed clinically as Combined Immune Deficiency with CD4 and B lymphopenia and markedly deficient IgG and increased IgM. The E525K mutation was not detected in our cohort. ConclusionsScreening for APDS in patients with recurrent respiratory tract infections with undefined antibody deficiency or combined immune deficiency with or without bronchiectasis is required. These patients need great attention to benefit from the available treatment. Further studies on the Egyptian population are recommended to increase the knowledge about the prevalence and phenotypic characterization of this disease in Egypt.

Development and application of two novel functional molecular markers of BADH2 in rice

Fragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase ( BADH2 ) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding. In this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 ( badh2-E2 ) or an 8-bp deletion and 3-bp variation in exon 7 ( badh2-E7 ). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2-E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F 2 population. Two functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future. How to cite: Li W, Zeng X, Li S, et al. Development and application of two novel functional molecular markers of BADH2 in rice. Electron J Biotechnol 2020;46. https://doi.org/10.1016/j.ejbt.2020.04.004 .

Detection of VT1 and VT2 genes in Escherichia coli isolated from Diarrhea Patients in AL-Anbar, Iraq using PCR Sequencing

Enteric-borne diarrheal illnesses are significant causes of human morbidity and mortality. Over 2 million diarrhea-related fatalities happen annually, especially among kids under the age of five. During the period extended from February 2019 to June 2019, A total of 196 stool locally specimens from diarrheal patients samples were collected from AL-Anbar city of AL-Ramadi hospital and AL-Fallujah hospital out of 196 samples diagnosis by biochemical test , morphology and selective chromogenic medium, found 80 samples of E. coli . The aim of the study detection of VT1 and VT2 genes in Escherichia coli isolated from Diarrhea patients by using PCR Sequencing. The result showed when diagnosis by PCR for Sanger Sequencing technique for VT1 and VT2 genes E. coli O157:H7,and E. coli O25b:H4 were positive for VT1gene ,and Escherichia coli O25b:H4 ,and Escherichia coli O18:H1 positive for VT2gene.

Development of a Rapid and Sensitive IDH1/2 Mutation Detection Method for Glial Tumors and a Comparative Mutation Analysis of 236 Glial Tumor Samples.

The presence of mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1/2) in glioma tumors is correlated with good prognosis upon standard-of-care treatment. Therefore, information on whether the glioma tumor has IDH1/2 mutations could be used in the correct diagnosis and management of glial tumors. The two most common techniques used to detect IDH1/2 mutations, immunohistochemistry (IHC) and Sanger sequencing, are prone to missing these mutations, especially if the tumor cells that carry the mutations constitute a small minority of the tumor itself. We developed and validated a rapid method (3-mismatch-amplification refractory mutation system [3m-ARMS]) that can be used for pre-, intra- and postoperative detection of the most common IDH1/2 mutations in glial tumors with high specificity and sensitivity. We also conducted a comprehensive IDH1/2 mutation analysis in 236 glial tumor samples comparing 3m-ARMS, IHC and Sanger sequencing. 3m-ARMS was optimized and validated for the specific and sensitive detection of the most common IDH1 and IDH2 mutations. We then analyzed 236 glial tumor samples for the presence of IDH1/2 mutations using 3m-ARMS, Sanger sequencing and IHC techniques. We then analyzed and compared the results, evaluating the diagnostic and screening potential of 3m-ARMS. Comparison of the three techniques used in the mutation analysis showed that 3m-ARMS-based IDH1/2 mutation detection was superior to IHC and Sanger sequencing-based IDH1/2 mutation detection in terms of accuracy, specificity and sensitivity, especially for tumor samples in which only a small minority of the cell population carried the mutation. 3m-ARMS could detect the presence of femtogram levels of IDH1/2 mutant DNA in DNA samples in which the mutant DNA-to-wild-type DNA ratio was as low as 1:100,000. Sanger sequencing and IHC-based methods have shortcomings when detecting mutations in glial tumors so can miss IDH1/2 mutations in glial tumors when used alone without proper modifications. 3m-ARMS-based mutation detection is fast and simple with potential for use as a diagnostic test for the majority of hot spot mutations in IDH1/2 genes. It can detect IDH1/2 mutations within an hour so can be adapted for intraoperative diagnosis.