Dot Blot Hybridization Research Articles

Protecting potato plants against PVX and PVY viral infections by the application of native and chemically modified legume proteins

Methylated soy and chickpea proteins (MSP and MCP, respectively), and 11S globulin (a soy protein fraction), are characterized by net positive charges and a more hydrophobic nature, nominating them as antiviral proteins. Under greenhouse conditions, potato plants (cv. Spunta) mechanically infected with potato virus Y (PVY) and potato virus X (PVX) were treated with MSP, MCP, and 11S globulin at different concentrations, after ten days of infection. The three tested substances inhibited the viral propagation, where the concentration 500 µg ml−1 produced the maximum antiviral action. This influence was further augmented by applying the material twice 10 and 20 days after the viral infection. Assessing the viral load by a double antibody sandwich ELISA (DAS-ELISA), dot-blot hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the viral inhibition, in the following order: MSP > MCP > 11S globulin. Bringing the virus in contact with MCP produced deformed TEM (transmission electron microscope) viral micrographs referring to a potential direct action of the substance on the virus. The tested materials' antiviral activity at 500 µg ml−1 was positively reflected on the growth and yield of PVY or PVX-infected potato plants cultivated under open field conditions in January. Considerably high increases in the tuber yield of either PVY-infected (68, 42, and 36%) or PVX-infected plants (52, 41 and 30%), were produced by the treatment with MSP, MCP and 11S (500 µg ml−1), respectively.

Biological and molecular characterization of bean bushy stunt virus, a novel bipartite begomovirus infecting common bean in northwestern Argentina.

Common bean plants (Phaseolus vulgaris L.) showing different virus-like symptoms were collected in northwestern Argentina. Dot-blot hybridization tests showed that the begomoviruses bean golden mosaic virus and tomato yellow vein streak virus were the most prevalent, but they also revealed the presence of unknown begomoviruses. The complete genome sequence of one of these unknown begomoviruses was determined. Sequence analysis showed that the virus is a typical New World begomovirus, for which the name "bean bushy stunt virus" (BBSV) is proposed. Biological assays based on biolistic inoculations showed that BBSV induced leaf roll and stunting symptoms similar to those observed in the field-collected common bean sample.

Comprehensive diversity assessment of walnut‐associated xanthomonads reveal the occurrence of distinct Xanthomonas arboricola lineages and of a new species (Xanthomonas euroxanthea) within the same tree

AbstractXanthomonas arboricola pv. juglandis (Xaj) is the aetiological agent of walnut diseases causing economic losses on walnut production worldwide. This phytopathogen is spread around the world where walnuts are produced and has a considerable genetic diversity. Using a comprehensive sampling methodology, focusing on factors that could influence the diversity of walnut‐colonizing Xaj in Portugal, this work provides new insights on xanthomonad populations on walnut. Genetic diversity was assessed by multilocus sequence analysis (MLSA) and dot blot hybridization patterns on 131 Xanthomonas isolates obtained from 64 walnut trees considering epidemiological metadata such as year of isolation, distinct bioclimatic regions, production regimes, and host‐related features. The results showed that the majority of isolates were split into 17 lineages of Xaj, while the other isolates clustered in four MLSA groups that did not include Xaj strains. These four groups were represented by three lineages of X. arboricola, and 11 lineages of Xanthomonas spp., including strains assigned to the recently proposed new species Xanthomonas euroxanthea. Furthermore, distinct Xaj, X. arboricola, and Xanthomonas spp. were isolated from the same walnut tree, suggesting possible genetic admixture within the same host. Phylogenetic analysis through geoBurst revealed the high diversity of these Xanthomonas spp. populations. Assessment of type III effector genes gave the indication that some Xanthomonas spp. strains were nonpathogenic on walnut, with the exception for X. euroxanthea CPBF 424. Altogether, these findings add to the thorough characterization of walnut‐associated xanthomonads in Portugal, providing a comprehensive snapshot of the current diversity that could contribute to risk assessment analysis and improve phytosanitary control.

Evaluación clínica de un nuevo método molecular para la detección de microorganismos multirresistentes

IntroductionThe main objective of this work is to carry out the clinical validation of the trial with the AMR Direct Flow Chip® starting from either nasal swabs, rectal swabs directly or from isolated strains to detect antibiotic resistance genes. MethodsWe developed the preclinical validation of the assay with 104 known bacterial isolates. A total of 210 nasal or rectal swab samples were analyzed. The AMR assay is based on multiplex PCR followed by reverse dot blot hybridization on DNA arrays fully automated by using the HS24 platform. The completion time of the full analysis is 3 hours. ResultsBoth the sensitivity and specificity of the preclinical assay were 100%, with the 104 samples correctly identified. In the clinical validation, the sensitivity was 100% and the specificity was between 100% in rectal swabs and 97% in nasal swabs. ConclusionsThe AMR Direct Flow Chip® is a rapid and effective assay for the detection of multidrug-resistant microorganisms from nasal and rectal swab samples.

Genetics background of β-thalassemia (3.5kb deletion) in Southern Thailand: Haplotype analysis using novel reverse dot blot hybridization.

β-thalassemia (β-thal) (3.5kb deletion or NC_000011.10:g.5224302-5227791del3490bp) is a common mutation in southern Thailand. This study aimed to determine genetic diversity in subjects with β-thal (3.5kb deletion) alleles and to ascertain the origin of this mutation using haplotype and phylogenetic analysis. The study was carried out on members of the southern Thai population, including 45 normal individuals, 116 heterozygous β-thal (3.5kb deletion) and one homozygous β-thal (3.5kb deletion). The 5'-haplotype in β-globin gene cluster was examined using newly developed reverse dot blot hybridization (RDB) and compared with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results showed 100% concordance between the haplotype patterns of these two methods. From a total of 324 chromosomes, nine haplotypes were segregated. Haplotype H2 (+ - - - -) was the predominant haplotype observed in all 118 β-thal (3.5kb deletion) chromosomes, which revealed a single origin. The phylogenetic tree demonstrated that β-thal (3.5kb deletion) has an older genetic defect in this region. Moreover, the developed RDB is simple, less time-consuming, inexpensive, and does not restriction enzyme digestion.

A non-invasive method for detecting mitochondrial tRNAThr15927G>A mutation

To explore the feasibility of detecting maternal hereditary mitochondrial tRNAThr15927G>A (m.15927G>A) mutation using buccal swabs. We performed sequence analysis of mitochondrial DNA in blood samples from 2070 cases of maternal hereditary mitochondrial disease in the First Affiliated Hospital of Wenzhou Medical University, and identified 3 patients with m.15927G>A mutation.Buccal swabs and blood samples were obtained from the 3 patients (mutation group) and 3 normal volunteers (control group).After extracting whole genomic DNA from all the samples, the DNA concentration and purity were analyzed.The PCR products were subjected to dot blot hybridization, Southern blot hybridization, and DNA sequencing analysis to verify the feasibility of detecting m.15927G>A mutation using buccal swabs. There was no significant difference in DNA concentration extracted from buccal swabs and blood samples in either the mutation group or the control group (P > 0.05), but the purity of manually extracted oral mucosa DNA was significantly lower than that of whole blood and oral mucosa DNA extracted using commercial kits (P < 0.05).Dot blot hybridization and Southern blot hybridization both yielded positive results in the control group but negative results in the mutation group.DNA sequencing identified m.15927G>A mutation in all the samples from the mutation group. Buccal swabs collection accurate is an accurate and sensitive method for the detection of m.15927G>A mutation.

In situ localization of citrus exocortis viroid RNA using an optimized RNAscope™ assay

Author(s): Stanton, Daniel; Harper, Scott J.; Cowell, Sarah J.; Brlansky, Ron | Abstract: Due to their small size, locating pathogenic RNAs, such as viroids, in plant tissue and cell organelles has been difficult. Viroids are small circular single-stranded RNA virus-like plant pathogens that reduce plant growth, vigor, and yield in economically important crops such as potato, tomato, hops and citrus. Viroid infections in plants are largely diagnosed by dot blot hybridization, PAGE-gels, or RT-PCR. Because traditional plant in situ hybridization studies for viroids are often limited by target signal amplification and specificity of binding, we examined the use of RNAscope™ (Advanced Cell Diagnostics Inc., Newark, CA), an in situ hybridization method that amplifies the signal of a single target, to study the cellular distribution of citrus exocortis viroid (CEVd). We found that after optimization, CEVd was localized in nuclei of infected cells as clearly distinguishable punctates visible with light microscopy at low magnification, suggesting that the RNAscope™ can be used to study viroids in situ.

Assessment of the Current Status of Potyviruses in Watermelon and Pumpkin Crops in Spain: Epidemiological Impact of Cultivated Plants and Mixed Infections.

Viral infections on cucurbit plants cause substantial quality and yield losses on their crops. The diseased plants can often be infected by multiple viruses, and their epidemiology may depend, in addition to the agro-ecological management practices, on the combination of these viral infections. Watermelon mosaic virus (WMV) is one of the most prevalent viruses in cucurbit crops, and Moroccan watermelon mosaic virus (MWMV) emerged as a related species that threatens these crops. The occurrence of WMV and MWMV was monitored in a total of 196 apical-leaf samples of watermelon and pumpkin plants that displayed mosaic symptoms. The samples were collected from 49 fields in three major cucurbit-producing areas in Spain (Castilla La-Mancha, Alicante, and Murcia) for three consecutive (2018–2020) seasons. A molecular hybridization dot-blot method revealed that WMV was mainly (53%) found in both cultivated plants, with an unadvertised occurrence of MWMV. To determine the extent of cultivated plant species and mixed infections on viral dynamics, two infectious cDNA clones were constructed from a WMV isolate (MeWM7), and an MWMV isolate (ZuM10). Based on the full-length genomes, both isolates were grouped phylogenetically with the Emergent and European clades, respectively. Five-cucurbit plant species were infected steadily with either WMV or MWMV cDNA clones, showing variations on symptom expressions. Furthermore, the viral load varied depending on the plant species and infection type. In single infections, the WMV isolate showed a higher viral load than the MWMV isolate in melon and pumpkin, and MWMV only showed higher viral load than the WMV isolate in zucchini plants. However, in mixed infections, the viral load of the WMV isolate was greater than MWMV isolate in melon, watermelon and zucchini, whereas MWMV isolate was markedly reduced in zucchini. These results suggest that the impaired distribution of MWMV in cucurbit crops may be due to the cultivated plant species, in addition to the high prevalence of WMV.

HPV16 AND EXPRESSION OF PROTEIN P16INK4A AND E7 ONCOPROTEIN IN COLORECTAL CARCINOMA.

ABSTRACT - BACKGROUND:Human papillomavirus (HPV) is the agent of the most prevalent sexually transmitted diseases in the world associated with cervix and anal canal cancer. The action of HPV on colorectal carcinogenesis is not yet established.OBJECTIVE: This research aimed to study the possible correlation between the presence of HPV16 and the gene expression of p16INK4a protein and HPV E7 oncoprotein and their levels in colorectal carcinoma tissue.METHODS: A retrospective case-control study of 79 patients with colorectal carcinoma was divided into two groups: HPV-positive and HPV-negative. The polymerase chain reaction was performed, in addition to dot-blot hybridization for HPV16 and HPV18. Colorectal tissue samples were also subjected to immunohistochemical study to assess the tissue level of E7 and p16INK4a proteins.RESULTS: HPV was identified in 36 (45.6%) cases. There was no significant difference between groups regarding gender (p=0.056), age (p=0.1), colic and/or rectal location (0.098), and presence of HPV. Gene expression of HPV E7 oncoprotein was present in 3.12% of cases (p=0.9), and p16INK4a protein expression was observed in 46.3% (p=0.27) of those selected with HPV detection.CONCLUSION: Gene expression and tissue levels of E7 oncoprotein and p16INK4a protein found in HPV-positive patients suggest the absence of HPV16 oncogenic activity in colorectal carcinoma.

Metabolic Engineering of Isoflavonoid Biosynthesis by Expressing Glycine max Isoflavone Synthase in Allium cepa L. for Genistein Production.

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants and play a vital role in promoting human health. Genetic engineering of the metabolite synthesis pathway has turned out to be an attractive approach for the production of various secondary metabolites. In our study, we attempted to produce the isoflavone genistein, a well-known health-promoting metabolite, in Allium cepa L. (onion) by introducing Glycine max Isoflavone synthase (GmIFS). The GmIFS gene was cloned into the pEarleyGate 102 HA vector and transformed into onion by Agrobacterium-mediated and biolistic methods. The presence of GmIFS in transgenic onion was confirmed by PCR, dot blot, and Southern hybridization. Analysis of the transgenic onion calli lines demonstrated that the expression of the GmIFS gene led to the production of isoflavone genistein in in vitro tissues. The biolistic stable transformed calli with transformation efficiency of 73% (62.65 nM/g FW) accumulated more genistein than the Agrobacterium stable transformed calli with transformation efficiency of 56% (42.5 nM/g FW). Overall, heterologous gene expression of GmIFS was demonstrated by modifying the secondary metabolite pathway in onion tissues for the production of isoflavone genistein that can boost up human health with its health-promoting properties.

Application of a dot blot hybridization assay for genotyping Streptococcus uberis from Brazilian dairy herds

Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to dot blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical dot blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.

Genomic and antibody-based assays for the detection of Indian strains of Macrobrachium rosenbergii nodavirus and extra small virus associated with white tail disease of Macrobrachium rosenbergii.

White tail disease (WTD) of cultured Macrobrachium rosenbergiiis caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Since both the viruses have small single strand RNA as genetic material with short generation time, they are more prone to mutations. Hence detection methods developed for one strain may be suboptimal for the detection of isolates from the different geographical locations. In the present study two new genomic based methods (RT-PCR and dot-blot hybridization) along with one immunological method (polyclonal antibodies based detection) were developed for the detection of Indian isolates of MrNV and XSV. Among genomic based methods, RT-PCR assay developed was most sensitive. Sensitivity of detection of RT-PCR was 1fg (both MrNV and XSV) of total RNA extracted from purified viral inoculum preparation. In case of WTD positive whole tissue total RNA, the limit of detection was 10fg for both MrNV and XSV. Dot-blot hybridization had a detection limit of 10pg and 0.1ng for MrNV and XSV respectively when RNA extracted from viral inoculum preparation was used; 0.1ng and 1ng when WTD positive whole tissue total RNA was used. Polyclonal antibodies against recombinant proteins (MrNV and XSV capsid) were synthesised. Western blotting and indirect ELISA revealed that the antibodies produced to be specific and highly sensitive. Recombinant protein (antigen) of MrNV and XSV capsid were detected at the dilution of 1:8000. However in case of infected prawn tissue sample, MrNV and XSV were detected at the dilution of 1:32,000 and 1:64,000 respectively. All methods developed are field applicable.

Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs Among Mycobacterium tuberculosis by a Reverse Dot Blot Hybridization Assay.

BackgroundReliable and timely determination of second-line drug resistance is essential for early initiation effective anti-tubercular treatment among multi-drug resistant (MDR) patients and blocking the spread of MDR and extensively drug-resistant tuberculosis. Molecular methods have the potency to provide accurate and rapid drug susceptibility results. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of fluoroquinolones (FQs), kanamycin (KN), amikacin (AMK), capreomycin (CPM) and second-line injectable drugs (SLIDs) in Mycobacterium tuberculosis.MethodsWe established and evaluated the accuracy of the RDBH assay by comparing to the phenotypic drug susceptibility testing (DST) and sequencing in 170 M. tuberculosis, of which 94 and 27 were respectively resistant to ofloxacin (OFX) and SLIDs.ResultsThe results show that, compared to phenotypic DST, the sensitivity and specificity of the RDBH assay for resistance detection were 63.8% and 100.0% for OFX, 60.0% and 100.0% for KN, 61.5% and 98.1% for AMK, 50.0% and 99.3% for CPM, and 55.6% and 100% for SLIDs, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 95.2% and 100.0% for OFX, 93.8% and 100.0% for SLIDs or KN (both based on mutations in rrs 1400 region and eis promoter), and 91.6% and 100.0% for AMK or CPM (both based on mutations in rrs 1400 region), respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples.ConclusionOur data suggested that compared to sequencing, the RDBH assay could serve as a rapid and reliable method for testing the resistance of M. tuberculosis against OFX and SLIDs, enabling early administration of appropriate treatment regimens among MDR tuberculosis patients.

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah, Saudi Arabia.

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.

First report of the spectrum of δ-globin gene mutations among women of reproductive age in Fujian area-Discrimination of δ-thalassemia, α-thalassemia, and Iron Deficiency Anemia.

BackgroundLow HbA2 level is an underlying of δ‐thalassemia, α‐thalassemia, and IDA. Interactions of these disorders can generate a wide spectrum of phenotype, which will pose diagnostic conundrum for clinical assessment, carrier screening, and genetic counseling.MethodsSubjects with HbA2 levels below 2.0% with normal or reduced hematological parameters were recruited for further investigation. δ‐globin gene mutations were identified by DNA sequencing of the HBD gene. Serum ferritin (SF) concentration was determined by the chemiluminescent microparticle immunoassay. The three common deletional α‐thalassemia (‐‐SEA/αα, ‐α3.7/αα, and ‐α4.2/αα) were detected using Gap‐PCR, detection of the point mutations in the three nondeletional α‐thalassemia (αCSα/αα,αQSα/αα,αWSα/αα), and the 17 common β‐thalassemia was performed using reverse dot blot hybridization (RDB).ResultsWe had characterized the δ‐globin gene mutations in 20 cases, revealing a frequency of 0.4% in the women of reproductive age (20/4 792). Two previously known mutations:‐77 T > C and −30 T > C and 3 novel δ‐globin gene defects: −44G > A,CD87C > T, and CD134T > A were found. In the selected cases, we also found 85 cases confirmed with (51.2%,85/166) IDA and 39 cases (23.5%,39/166) with common α‐thalassemia. Subjects with δ‐thalassemia had statistically higher levels of Hb, MCV, and MCH compared with other two groups, whereas statistically lower levels of RDW were seen in δ‐thalassemia group. What's more, statistically higher levels of SF were seen in δ‐thalassemia group, compared with IDA groups.ConclusionWe reported the spectrum of δ‐thalassemia mutations for the first time with the frequency of 0.4% among women of reproductive age in Fujian area and found that −77T > C mutation was the most common mutation, followed by −30T > C mutation. What's more, 3 novel δ‐globin gene defects: −44G > A,CD87C > T and CD134T > A were found. A thorough analysis of the hematological, electrophoretic characterization, and the level of SF was needed to suspect and further investigate the existence of IDA, α‐thalassemia, and δ‐thalassemia.

Trinucleotide Repeat-Targeting dCas9 as a Therapeutic Strategy for Fuchs' Endothelial Corneal Dystrophy.

PurposeFuchs’ endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation. Seventy percent of cases are caused by an intronic CTG triplet repeat expansion in the TCF4 gene that results in accumulation of pathogenic expanded CUG repeat RNA (CUGexp) as nuclear foci in corneal endothelium. A catalytically dead Cas9 (dCas9) can serve as an effective guide to target genomic DNA or RNA transcripts. Here, we examined the utility of the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 system to effectively target and reduce CUGexp.MethodsWe delivered dCas9 and repeat-targeting single guide RNA (sgRNA) expression plasmids to patient-derived endothelial cells using lipofection or lentiviral transduction. We used fluorescence in situ hybridization (FISH) and RNA dot-blot hybridization to quantify CUGexp foci and repeat RNA levels, respectively. TCF4 expression levels were assessed using quantitative PCR (qPCR).ResultsUsing FISH, we found that expression of both dCas9 and a (CAG)n sgRNA complementary to CUGexp are necessary to reduce foci. We observed a reduction in percentage of cells with foci from 59% to 5.6% and number of foci per 100 cells from 73.4 to 7.45 (P < 0.001) in cells stably expressing dCas9-(CAG)n sgRNA but saw no decrease in cells expressing dCas9-(CUG)n sgRNA or nontargeting control sgRNA. In cells with dCas9-(CAG)n sgRNA, we detected a reduction in CUGexp RNA by dot-blot without any reduction in TCF4 mRNA levels using qPCR.ConclusionsUsing CRISPR-dCas9 to target the trinucleotide repeat is a promising treatment for FECD contingent on effective in vivo delivery.Translational RelevanceThis work advances a gene therapy for a common age-related degenerative disorder.

Alpha thalassemia genotypes in Kuwait

BackgroundThe frequency of the alpha thalassemia trait is approximately 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of patients who were referred for molecular diagnosis over a 20-year period.MethodsThis is a retrospective study of the α-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was performed by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot blot hybridization (Vienna Lab Strip Assay).ResultsFour hundred samples were characterized and analyzed from individuals aged < 1 month to 80 years, with a median of 6 years from 283 unrelated families. Most (90.8%) were Kuwaiti nationals. The commonest genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3% of the samples, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. PA-1 was therefore the most frequent allele (0.59). The frequency of the α0 (−-MED) allele was 0.017. Rare alleles that were found in very low frequencies included α0 (−-FIL) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria.ConclusionThere is a wide variety of alpha thalassemia alleles among Kuwaitis, but nondeletional PA-1 is by far the most common cause of the moderate to severe HbH (β4 tetramer) disease phenotype. The α0 (−MED) allele is also encountered, which has implications for premarital counseling, especially for the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

Development of a novel polyprobe for simultaneous detection of six viruses infecting stone and pome fruits

A biotin-labeled, non-isotopic, novel polyprobe was developed for the simultaneous detection of six viruses viz. apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV), cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV) and plum pox virus (PPV) infecting stone and pome fruit trees through dot-blot hybridization assay. The sensitivity of the polyprobe was checked by serial dilutions of total RNA extracted from the tissues of infected trees. ACLSV was detected up to a dilution of 5-5, whereas ApMV, ASGV, CVA, PPV and PNRSV up to 5-4. The developed assay was validated following testing a total of 45 symptomatic leaf samples collected from different geographical regions of Jammu and Kashmir (India), and the presence of the viruses was further confirmed by RT-PCR and sequencing. The polyprobe, designed for performing molecular hybridization assay can be developed quickly and avoid the tedious transformation and cloning procedures. Apart from simultaneously detecting viruses in stone and pome fruit trees, it holds great potential for virus indexing programmes to ascertain the supply of virus-free plant materials to the growers.

Characterization of m6A in mouse ovary and testis

Characterization of m6A in mouse ovary and testis

Genotype Analysis of Pregnant Women with α- and β- Thalassemia in Fuzhou Area of Fujian Province in China

To analyze the genotype of pregnant women with α- and β- thalassemia in Fuzhou area of Fujian province in China. Blood routine examination and hemoglobin electrophoresis were performed for pregnant women, and positive samples were examined by gap polymerase chain reaction and reverse dot blot hybridization. 412 cases were diagnosed as α-thalassemia (63.9%); 201 cases were diagnosed as β-thalassemia (31.2%); 32 cases were diagnosed as α and β-composite thalassemia. There were 12 genotypes in α-thalassemia, whose major genotypes were --SEA/αα, α3.7/αα, -α4.2/αα and αQSα/αα, with carrying rate of 64.32%, 20.14%, 7.77% and 1.94%, respectively. There were 10 genotypes in β- thalassemia, whose major genotypes were CD41-42/N, CD17/N, IVS-II-654/N and -28/N, with carrying rate of 30.84%, 27.86%, 15.92% and 10.45%, respectively. There were 9 genotypes in α and β-composite thalassemia, whose major genotypes were --SEA/αα composited CD41-42/N, -α3.7/αα composited CD41-42/N, --SEA/αα composited CD17/N, with carrying rate of 18.75%, 15.62%, 15.62% respectively. The major genotypes of pregnant women with α- and β- thalassemia in Fuzhou area of Fujian province in China are --SEA/αα, α3.7/αα, CD41-42/N and CD17/N. Thalassemia screening and prenatal gene diagnosis should be strengthened in Fuzhou area of Fujian province in China.