Blot Hybridization Technique Research Articles

Long-Read Sequencing Identified a Large Novel δ/β-Globin Gene Deletion in a Chinese Family

Objective. Increasingly rare thalassemia has been identified with the advanced use of long-read sequencing based on long-read technology. Here, we aim to present a novel δ/β-globin gene deletion identified by long-read sequencing technology. Methods. Enrolled in this study was a family from the Quanzhou region of Southeast China. Routine blood analysis and hemoglobin (Hb) capillary electrophoresis were used for hematological screening. Genetic testing for common α- and β-thalassemia was carried out using the reverse dot blot hybridization technique. Long-read sequencing was performed to detect rare globin gene variants. Specific gap-polymerase chain reaction (gap-PCR) and/or Sanger sequencing were further used to verify the detected variants. Results. None of the common α- and β-thalassemia mutations or deletions were observed in the family. However, decreased levels of MCV, MCH, and abnormal Hb bands were observed in the family members, who were suspected as rare thalassemia carriers. Further, long-read sequencing demonstrated a large novel 7.414 kb deletion NG_000007.3:g.63511_70924del partially cover HBB and HBD globin genes causing delta-beta fusion gene in the proband. Parental verification indicated that the deletion was inherited from the proband’s father, while none of the globin gene variants were observed in the proband’s mother. In addition, the novel δ/β-globin gene deletion was further verified by gap-PCR and Sanger sequencing. Conclusion. In this study, we first present a large novel δ/β-globin gene deletion in a Chinese family using long-read sequencing, which may cause δβ-thalassemia. This study further enhances that long-read sequencing would be applied as a sharp tool for detecting rare and novel globin gene variants.

A microfluidic chip-based multiplex PCR-reverse dot blot hybridization technique for rapid detection of enteropathogenic bacteria

Diarrhea caused by enteropathogenic bacteria is a major public health issue worldwide, especially in developing countries. In this study, a microfluidic chip-based multiplex polymerase chain reaction (PCR)-reverse dot blot hybridization technology for the rapid and simultaneous detection of 11 enteropathogenic bacteria was developed and the entire process was completed within 3–4 h. The specificity of this method was analyzed using 11 types of pure target bacterial colonies and another 7 types of pure bacterial colonies, and its sensitivity was evaluated with the serial 10-fold dilution of 11 types of pure target bacterial colonies. The detection limit of this method was as low as 103–102 CFU/mL, and it exhibited high specificity for enteropathogenic bacteria. A total of 60 clinical diarrheal fecal samples were detected using this method, the results of which were compared with those of the conventional reference method, which resulted in a positive coincident rate of 100% and a negative coincident rate of 93.75%. Based on the findings, it could be concluded that multiplex PCR-reverse dot blot hybridization based on the microfluidic chip is a rapid, economical, sensitive, specific, and high-throughput method for detecting enteropathogenic bacteria.

Analysis of Gene Detection and Hematological Phenotype of Thalassemia

To investigate gene mutation types of thalassemia in the population of this area and the relationship between hematological phenotype and age in β-thalassemia. 1 351 suspected cases of thalassemia were detected in the First Affiliated Hospital of Chengdu Medical College from June 2017 to June 2021. PCR-reverse dot blot hybridization(PCR-RDB) technique was used to detect the common α and β thalassemia gene types, then the data of MCV, MCH, MCHC and Hb of confirmed cases were collected. The heterozygotes of β-thalassemia were divided into 0-18 year group, 19-50 year group and >50 year group according to age, and the differences of hematological phenotypes among different groups were compared. Among the detected 1 351 samples， 523(38.71%) cases were diagnosed as thalassemia, 13 genotypes were detected in 260 cases (19.25%) with α-thalassemia; and 12 genotypes were detected in 252 cases (18.65%) with β-thalassemia; 9 genotypes were detected in 11 cases with αβ thalassemia. It was found that MCV and MCH were increased significantly in 0-18 year group, 19-50 year group and >50 year group, MCHC was highest in 0-18 year group, and Hb was the lowest in the >50 year group, and the difference was statistically significant（P<0.05）. In this area, --SEA/αα was the major genotype of α-thalassemia, and CD41-42 /N and IVS-II-654/N were the major genotypes of β-thalassemia. Morever, MCV and MCH were the lowest in the 0-18 year old group and the highest in the >50 years old group in heterozygotes of β-thalassemia. The influence of age on hematological parameters in the primary screening of thalassemia provides a certain reference value in clinical diagnosis and treatment.

Designing a Sequence-Based Method for Identifying 14 High-Risk Carcinogenic HPV Types in Multiple Infections.

HPV tests have significant drawbacks in terms of detecting and differentiating types of the virus. PCR techniques provide timely and necessary results for patient care with high quality, sensitivity, and reasonable cost. The sensitivity of PCR depends on the primers. In this study, a method was designed that exploited PCR with designed primers (ScTd) by changing the annealing temperature (Ta) along with Sanger sequencing for pap smear samples. Sanger sequencing has confirmed that ScTd primers have a relative differentiation power using PCR. The primers caused a relative differentiation by PCR. In the pap smear sample 22 with contamination of types 16, 31, and 45, confirmed by dot blot hybridization, type 16 was not amplified at the specific Ta. Moreover, the band was observed at low Ta. Sanger sequencing showed that type 16 was detected instead of type 52. Sequencing the heterozygous bands in multiple infections also led to the identification of different types. Moreover, with a combination of 7 pairs of primers, HPV types can be detected in multiple infections by PCR. As compared with the clinical dot blot hybridization technique, the utilization of complementary PCR and sequencing methods with designed primers can provide a higher positive predictive value in the detection of high-risk types.

Thalassemia Gene Detection Results and Application Value of Hematological Indexes Among Pregnant Women in Xindu District of Chengdu City

To analyze the genotype characteristics of α- and β-thalassemia and the diagnostic value of hematological indexes in pregnant women in Xindu District of Chengdu. The blood routine parameters(MCV) <80 fl and (or) (MCH) <27 pg and hemoglobin electrophoresis were used to screen the pregnant women, PCR-reverse dot blot hybridization(PCR-RDB) technique was used to detect the common α- and β-thalassemia gene types in the primary screening positive population. The husbands of the diagnosed pregnant women were recalled for gene testing, and the highly suspected patients were checked by gene sequencing. Among the 7 049 pregnant women, 1 740(24.68%) cases were positive for primary screening. 180 patients were diagnosed as thalassemia gene positive, among them, 94 cases (52.22%) of α-thalassemia were detected and six genotypes were found, in which --SEA /αα genotype was the highest (58 cases, 61.70%); 82 cases (45.56%) of β-thalassemia were detected and ten genotypes were found while CD41-42/N and CD17/N genotypes were the most common; there were 4 cases(2.22%) with α combined with β-thalassemia. Through clinical follow-up survey, there were 4 couples with the same type of thalassemia, one of them was induced labor after diagnosis of hemoglobin H disease. Receiver operating curve (ROC curve) was used to analyze the diagnostic value of hematological parameters in thalassemia positive pregnant women. The results showed that AUC(HBA2)<AUC(MCHC)<AUC(RDW-SD)<AUC(MCH)<AUC(MCV) (P<0.01). The most common genotypes of α- and β-thalassemia in pregnant women in Xindu District of Chengdu were --SEA /αα, CD41-42/N, CD17/N. The blood routine indicators (HBA2、RDW-SD、MCHC、MCH、MCV) have high diagnostic value for screening of thalassemia.

Genotypes of thalassemia in children: an analysis of 30 417 cases.

To investigate the distribution of genotypes of thalassemia in children in Guangxi, China. A total of 30 417 children with positive results of thalassemia screening in the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region from January 2011 to December 2019 were enrolled. Single-tube multiplex PCR, agarose gel electrophoresis, and reverse dot blot hybridization technique were used for the detection of common α- and β-thalassemia genes. Gap-PCR or gene sequence analysis was performed for 2 703 children suspected of rare thalassemia. Among the 30 417 children with positive results of thalassemia screening, 23 214 (76.32%) were diagnosed with thalassemia, and the detection rates of α-thalassemia, β-thalassemia, and α-thalassemia with β-thalassemia were 47.77%, 23.75%, and 4.80% respectively. A total of 13 types of α-thalassemia alleles (18 480 alleles in total) were detected, mainly --SEA (54.98%), including seven rare alleles, i.e., --THAI (0.43%), HKαα (0.02%), -α30 (0.01%), -α1.0 (0.01%), -α2.4 (0.01%), -α21.9 (0.01%), and HBA2:C272-279 del (0.01%). A total of 17 types of β-thalassemia alleles (9 168 alleles in total) were detected, mainly CD41-42 (47.79%), followed by CD17 (25.53%), including three rare alleles, i.e., IVS-II-5 (0.02%), IVS-I-2 (0.01%), and Gγ(Aγδβ)0 (0.01%). A total of 37 genotypes were detected in 14 531 children with α-thalassemia, among which the most common 6 genotypes were --SEA/αα (52.20%), -α3.7/αα (13.24%), αCSα/αα (7.52%), -α4.2 (6.06%), --SEA/-α3.7 (5.91%), and αWSα/αα (3.41%), accounting for 88.34%. A total of 49 genotypes were detected in 7 223 children with β-thalassemia, among which the most common 6 genotypes were CD41-42/βN (45.81%), CD17/βN (24.30%), IVS-II-654/βN (7.49%), -28/βN (5.62%), CD71-72/βN (4.42%), and CD26/βN (3.94%), accounting for 91.13%. A total of 137 genotypes were detected in 1 460 children with both α- and β-thalassemia, mainly --SEA/αα combined with CD41-42/βN (14.17%) and CD17/βN (8.35%). A total of 2 050 children were diagnosed with hemoglobin H disease (α0/α+), among whom 134 had β-thalassemia heterozygote and 12 had Bart hydrops fetalis syndrome (--SEA/--SEA); 355 children were diagnosed with β-thalassemia double heterozygote, and 128 were diagnosed with β-thalassemia homozygote, including 93 children with α-thalassemia. There are diverse gene mutations and rich genotypes of thalassemia among children in Guangxi, and α-thalassemia is more common, with --SEA/αα as the major genotype. There is a high proportion of children with both α- and β-thalassemia, and there are relatively high incidence rates of β-thalassemia double heterozygote and homozygote (intermedia and major).

Antiviral Action of Native and Methylated Lactoferrin and β-Lactoglobulin against Potato Virus Y (PVY) Infected into Potato Plants Grown in an Open Field.

Potato plants are liable to PVY infection without efficient control. Therefore, they were cultivated under greenhouse and open field conditions, artificially infected with PVY and then treated after 15 days of infection with native lactoferrin (LF) and native β-lactoglobulin (BL) and their esterified forms, MLF (methylated lactoferrin) and BLM (methylated β-lactoglobulin) to test the efficiency of this approach. Viral replication was inhibited by the applied substances, particularly the methylated forms, in a concentration-dependent manner, where the concentration of 500 μg·mL−1 was sufficient for plant protection against the PVY infection. An open field experiment showed that one single application of the antiviral substance was enough for maximum inhibitory action against PVY. The modified milk proteins induced higher inhibitory action on PVY virus replication in the plants, compared to their native forms, which was reflected by potato growth and yield. Using the dot blot hybridization and RT-PCR techniques to detect PVY in the experimental plants showed the supremacy of native and esterified LF in inhibiting the targeted virus. The generally observed scanning electronic microscopy (SEM) structural deformations and irregular appearance in PVY particles when treated with MLF and BLM revealed their direct action. BLM, MLF and LF are efficient antiviral agents against PVY. They can not only abolish the observed PVY-induced reduction in potato growth and tuber yield, but also further increase them to higher levels than negative control.

Remarkable variation of ribosomal DNA organization and copy number in gnetophytes, a distinct lineage of gymnosperms.

IntroductionGnetophytes, comprising the genera Ephedra, Gnetum and Welwitschia, are an understudied, enigmatic lineage of gymnosperms with a controversial phylogenetic relationship to other seed plants. Here we examined the organization of ribosomal DNA (rDNA) across representative species.MethodsWe applied high-throughput sequencing approaches to isolate and reconstruct rDNA units and to determine their intragenomic homogeneity. In addition, fluorescent in situ hybridization and Southern blot hybridization techniques were used to reveal the chromosome and genomic organization of rDNA.Key resultsThe 5S and 35S rRNA genes were separate (S-type) in Gnetum montanum, Gnetum gnemon and Welwitschia mirabilis and linked (L-type) in Ephedra altissima. There was considerable variability in 5S rDNA abundance, ranging from as few as ~4000 (W. mirabilis) to >100 000 (G. montanum) copies. A similar large variation was also observed in 5S rDNA locus numbers (two to 16 sites per diploid cell). 5S rRNA pseudogenes were interspersed between functional genes forming a single unit in E. altissima and G. montanum. Their copy number was comparable or even higher than that of functional 5S rRNA genes. In E. altissima internal transcribed spacers of 35S rDNA were long and intrinsically repetitive while in G. montanum and W. mirabilis they were short without the subrepeats.ConclusionsGnetophytes are distinct from other gymnosperms and angiosperms as they display surprisingly large variability in rDNA organization and rDNA copy and locus numbers between genera, with no relationship between copy numbers and genome sizes apparent. Concerted evolution of 5S rDNA units seems to have led to the amplification of 5S pseudogenes in both G. montanum and E. altissima. Evolutionary patterns of rDNA show both gymnosperm and angiosperm features underlining the diversity of the group.

Copy Number of Human Ribosomal Genes With Aging: Unchanged Mean, but Narrowed Range and Decreased Variance in Elderly Group.

Introduction: The multi-copied genes coding for the human 18, 5.8, and 28S ribosomal RNA (rRNA) are located in five pairs of acrocentric chromosomes forming so-called rDNA. Human genome contains unmethylated, slightly methylated, and hypermethylated copies of rDNA. The major research question: What is the rDNA copy number (rDNA CN) and the content of hypermethylated rDNA as a function of age?Materials and Methods: We determined the rDNA CN in the blood leukocyte genomes of 651 subjects aged 17 to 91 years. The subjects were divided into two subgroups: “elderly” group (E-group, N = 126) – individuals over 72 years of age (the age of the population’s mean lifetime for Russia) and “non-elderly” group (NE-group, N = 525). The hypermethylated rDNA content was determined in the 40 DNA samples from the each group. The change in rDNA during replicative cell senescence was studied for the cultured skin fibroblast lines of five subjects from NE-group. Non-radioactive quantitative dot- and blot-hybridization techniques (NQH) were applied.Results: In the subjects from the E-group the mean rDNA CN was the same, but the range of variation was narrower compared to the NE-group: a range of 272 to 541 copies in E-group vs. 200 to 711 copies in NE-group. Unlike NE-group, the E-group genomes contained almost no hypermethylated rDNA copies. A case study of cultured skin fibroblasts from five subjects has shown that during the replicative senescence the genome lost hypermethylated rDNA copies only.Conclusion: In the elderly group, the mean rDNA CN is the same, but the range of variation is narrower compared with the younger subjects. During replicative senescence, the human fibroblast genome loses hypermethylated copies of rDNA. Two hypotheses were put forward: (1) individuals with either very low or very high rDNA content in their genomes do not survive till the age of the population’s mean lifetime; and/or (2) during the aging, the human genome eliminates hypermethylated copies of rDNA.

Diagnostic Parameters of Northern Blot Hybridization Technique for Detection of Citrus Viroids in Field-grown Plants

Diagnostic Parameters of Northern Blot Hybridization Technique for Detection of Citrus Viroids in Field-grown Plants

Piroplasms in brown hyaenas (Parahyaena brunnea) and spotted hyaenas (Crocuta crocuta) in Namibia and South Africa are closely related to Babesia lengau.

The objective of our study was identification and molecular characterization of piroplasms and rickettsias occurring in brown (Parahyaena brunnea) and spotted hyaenas (Crocuta crocuta) from various localities in Namibia and South Africa. Whole blood (n=59) and skin (n=3) specimens from brown (n=15) and spotted hyaenas (n=47) were screened for the presence of Babesia, Theileria, Ehrlichia and Anaplasma species using the reverse line blot (RLB) hybridization technique. PCR products of 52/62 (83.9%) of the specimens hybridized only with the Theileria/Babesia genus-specific probes and not with any of the species-specific probes, suggesting the presence of a novel species or variant of a species. No Ehrlichia and/or Anaplasma species DNA could be detected. A parasite 18S ribosomal RNA gene of brown (n=3) and spotted hyaena (n=6) specimens was subsequently amplified and cloned, and the recombinants were sequenced. Homologous sequence searches of databases indicated that the obtained sequences were most closely related to Babesia lengau, originally described from cheetahs (Acinonyx jubatus). Observed sequence similarities were subsequently confirmed by phylogenetic analyses which showed that the obtained hyaena sequences formed a monophyletic group with B. lengau, B abesia conradae and sequences previously isolated from humans and wildlife in the western USA. Within the B. lengau clade, the obtained sequences and the published B. lengau sequences were grouped into six distinct groups, of which groups I to V represented novel B. lengau genotypes and/or gene variants. We suggest that these genotypes cannot be classified as new Babesia species, but rather as variants of B. lengau. This is the first report of occurrence of piroplasms in brown hyaenas.

Northern Blotting Techniques for Small RNAs.

Cells have evolved intricate RNA-directed mechanisms that destroy viruses, silence transposons, and regulate gene expression. These nucleic acid surveillance and gene silencing mechanisms rely upon the selective base-pairing of ~19-25 nt small RNAs to complementary RNA targets. This chapter describes northern blot hybridization techniques for the detection of such small RNAs. Blots spiked with synthetic standards are used to illustrate the detection specificity and sensitivity of DNA oligonucleotide probes. Known endogenous small RNAs are then analyzed in samples prepared from several model plants, including Arabidopsis thaliana, Nicotiana benthamiana, Oryza sativa, Zea mays, and Physcomitrella patens, as well as from the animals Drosophila melanogaster and Mus musculus. Finally, the value of northern blotting for dissecting small RNA biogenesis is shown using an example of virus infection in A. thaliana.

The widespread occurrence of tRNA-derived fragments in Saccharomyces cerevisiae.

Short RNAs derived from the cleavage of tRNA molecules are observed in most organisms. Their occurrence seems to be induced by stress conditions, but still little is known about their biogenesis and functions. We find that the recovery of tRNA fragments depends on the RNA isolation method. Using an optimized RNA extraction protocol and northern blot hybridization technique, we show that the tRNA‐derived fragments in yeast are widespread in 12 different growth conditions. We did not observe significant stress‐dependent changes in the amounts of tRNA fragments pool. Instead, we show the differential processing of almost all individual tRNAs. We also provide evidence that 3′‐part‐derived tRNA fragments are as abundant as the 5′‐ one in Saccharomyces cerevisiae. The resulting set of S. cerevisiae tRNA fragments provides a robust basis for further experimental studies on biological functions of tRFs.

Genetic Transformation of Dendrobium 'Sonia Earsakul' with Antisense Carica papaya ACO1 Gene

&lt;p&gt;&lt;em&gt;Dendrobium&lt;/em&gt; orchid is one of the major export cut flowers not only in Thailand but also for several tropical countries. However, the production of ethylene by their flowers causes a shorter vase life. Flowers that contained lower levels of ethylene usually exhibited delayed senescence and consequently prolonged vase life. The transfer of antisense &lt;em&gt;ACC oxidase (ACO)&lt;/em&gt; gene into orchid, in theory, may leads to decreased ethylene production because this gene can down regulates the ethylene biosynthesis pathway. This study focuses on the transformation and the existence and expression of the antisense &lt;em&gt;ACO&lt;/em&gt;1 gene from papaya, namely (&lt;em&gt;CP-ACO&lt;/em&gt;1), which was transferred in to &lt;em&gt;Dendrobium&lt;/em&gt; 'Sonia Earsakul'. The successful stable transformation event obtained and the existence of the transferred gene was determined using PCR, dot blot hybridization and Southern blot hybridization techniques. The results revealed that antisense &lt;em&gt;CP-ACO&lt;/em&gt;1 and &lt;em&gt;hygromycin phosphotransferase (hpt)&lt;/em&gt; gene existed in all transgenic lines confirmed by PCR technique. The genomic dot blot confirmed the incorporation of the transgene in transgenic plant genome. Southern blot hybridization revealed the existed of one to four sets of the gene in transgenic lines. The expression of antisense &lt;em&gt;CP-ACO&lt;/em&gt;1 gene was analyzed through the level of ACO enzyme activity and ethylene production in transgenic orchid. All of the transgenic lines had lower ACO enzyme activity and lower ethylene production than that of the non-transgenic orchid plants.&lt;strong&gt; &lt;/strong&gt;&lt;/p&gt; &lt;p&gt; &lt;/p&gt;

XmnI polymorphism: Relation to β-thalassemia phenotype and genotype in Egyptian Children

Backgroundβ-Globin mutations with Xmn1 site might be associated with elevated HbF expression which may in turn ameliorate the severity of β-thalassemia phenotype. Aim of the studyTo investigate the frequency of −158 (C>T) XmnI polymorphism among Egyptian Children and young adults with β-thalassemia, to examine the relationship between XmnI polymorphism and β-thalassemia genotypes and phenotypes and to assess the possible relation of XmnI polymorphism and response to hydroxyurea (Hu) therapy. Patients and methodsSeventy-two β-thalassemia patients (37 females; M/F ratio 0.95) with a mean age of 7.53±6.99 were included. Laboratory investigations included Complete blood count (CBC), Hb electrophoresis by high performance liquid chromatography (HPLC), β-thalassemia mutation identification by the reverse dot blot hybridization technique (RDB) and detection of XmnlGg polymorphism by RFLP. ResultsThe frequency of positive heterozygote XmnI gene polymorphism was 8.3%. Eighty-three percent of XmnIGγ+/− patients were never transfused (p=0.001) and had higher total hemoglobin compared to XmnIGγ−/− (p=0.01); while mean HbF was higher among XmnIGγ+/− patients compared to the other group but the difference was marginally insignificant (p=0.06). β-Thalassemia mutation IVS II-1 showed relatively higher XmnI polymorphism frequency (50%) and followed by its frequency among 10 undefined β-thalassemia mutations which was 20%. The frequency of positive heterozygote XmnI gene polymorphism was 11.6% among the TI group vs. 3.5% among the TM group (p=0.4). Among 20 cases who received HU; 5/14 responders vs. 1/6 none responder had positive heterozygote XmnI gene polymorphism (p=1.0). Conclusions and recommendationsIn conclusion, molecular determination of genetic markers in childhood will help to identify phenotypes of our patients and to avoid over or under treatment strategies. Further prospective studies concerning the genetic markers that could predict the response to hemoglobin F inducers like hydroxyurea are highly recommended.

Alpha-thalassemia mutations in adana province, southern Turkey: genotype-phenotype correlation.

To look over the distribution of the mutations in a large series from Adana province, Southern Turkey, and determine the genotype-phenotype correlation of the frequent mutations. Among the 2500 individuals with mild or moderate anemia, microcytosis, and normal iron levels that were referred to our Genetic Diagnosis Center, a population consisting of 539 individuals were included in the study and tested for alpha-thalassemia mutations by using reverse dot blot hybridization technique. Twelve different mutations were detected in 539 patients. Among the 12 different mutations found, the most frequent mutations were the -α(3.7) (63.3%), --(MED) (11.7%), --(20.5) (10.7%), α2(IVS1(-5nt)) (3.9%), and α2(polyA-2) (3.5%). The most frequent genotypes were -α(3.7)/αα (35.8%), -α(3.7)/-α(3.7)(18.9%), -(20.5)/αα (11.5%), and --(MED)/αα (10.4%), respectively. There were statistically significant differences in hematological findings between -α(3.7)/-α(3.7) and --(MED)/αα, even though both have two mutated genes in the genotype. Our results show that alpha-thalassemia mutations are highly heterogeneous as well as deletional and -α(3.7) single gene deletion is particularly prevalent at Adana province in agreement to other studies from Turkey.

A Distinct Strain of Chickpea chlorotic dwarf virus Infecting Pepper in Oman.

During a field survey in 2011, pepper (Capsicum annum) plants showing symptoms suggestive of geminivirus infection were observed in three fields in the Al-Sharqiya region of Oman. Symptoms observed included upward leaf curling leading to cupping and stunting with 15 to 25% disease incidence in surveyed fields. Total DNA was extracted from the leaves of seven symptomatic plants and subjected to rolling circle amplification (RCA). The RCA product was digested with several restriction endonucleases to obtain unit length of ~2.6 to 2.8, typical of geminivirus. Out of seven samples, only four yielded a product of ~2.6 kb in size by KpnI digestion. The fragments were cloned in pUC19 and sequenced. The partial sequences of four isolates were >95% identical to each other at the nucleotide (nt) level and thus only one isolate (P-25) was fully sequenced, determined to be 2,572 nt in length, and its sequence deposited in GenBank (KF111683). The P-25 sequence showed a genome organization typical of a mastrevirus, with four open reading frames (ORFs), two in virion-sense and two in complementary-sense. The virion and complementary-sense ORFs were separated by a long intergenic region, containing a predicted hairpin structure with the nonanucleotide sequence (TAATATTAC) in the loop, and a short intergenic region. An initial comparison to all sequences in the NCBI database using BlastN showed the isolate to have the highest level of sequence identity with isolates of the dicot-infecting mastrevirus Chickpea chlorotic dwarf virus (CpCDV). Subsequent alignments of all available CpCDV isolates using the species demarcation tool (2) showed the isolate P-25 to share between 83.6 and 90.3% identity to isolates of CpCDV available in databases, with the highest (90.3%) to CpCDV strain A originating from Syria (FR687959) (3). Amino acid sequence comparison showed that the predicted proteins encoded by the four ORFs of P-25 (coat protein [CP], movement protein [MP], replication associated protein A [RepA], and RepB) share 91.5, 88.2, 89.1, and 90.8% amino acid sequence identity, respectively, with the homologous proteins of the CpCDV isolate from Syria. Based on the recently revised mastreviruses species and strain demarcation criteria (78 and 94% whole genome nt identity, respectively) proposed by Muhire et al. (2), the results indicate that isolate P-25 represents a newly identified strain (strain F) of CpCDV. The presence of CpCDV in symptomatic pepper plants was further confirmed by Southern blot hybridization technique using digoxygenin (DIG) labeled probe prepared from CpCDV isolate P-25. The genus Mastrevirus consists of geminiviruses with single component genomes that are transmitted by leafhoppers. Mastreviruses have so far only been identified in the Old World and infect either monocotyledonous or dicotyledonous plants (1). To our knowledge, this is the first report of a mastrevirus on the Arabian Peninsula and the first record of pepper as host of CpCDV. Recently, several begomoviruses of diverse geographic origins have been found infecting vegetable crops in Oman. The propensity of geminiviruses to evolve through recombination may lead to evolution of recombinant CpCDV with new host adaptability. Due to extensive agricultural/travel links of Oman with rest of the world, there exists high probability for the spread of this virus.

Prevalence of sulfadoxine–pyrimethamine resistance-associated mutations in dhfr and dhps genes of Plasmodium falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia

Ethiopia changed the first-line anti-malarial drug for uncomplicated Plasmodium falciparum malaria from sulfadoxine–pyrimethamine (SP) to Coartem® in 2004 following nation-wide assessment of the efficacy of both drugs in 2003. This study was conducted to assess the prevalence of sulfadoxine–pyrimethamine resistance-associated mutations in dhfr and dhps genes of P. falciparum three years after SP withdrawal in Bahir Dar, Northwest Ethiopia. A total of 165 blood spot samples were collected from patients infected with P. falciparum in Bahir Dar Health Center in 2005 (n=78) and 2008 (n=87) using Whatman (3M) filter papers. The three dhfr codons (dhfr108, dhfr 51 and dhfr 59) and the two dhps codons (dhfr 437 and 540) which are believed to determine SP resistance were detected by using nested PCR-based dot blot-hybridization technique. In dhfr, only the dhfr59Arg mutant-type showed statistically significant reduction from 80.3% in 2005 to 56.4% in 2008 (p<0.01) with a significant increase of the wild type dhfr59Cys haplotypes from 4.9% in 2005 to 29.5% in 2008 (p<0.01). The double mutants dhfr108Asn/51Ile were detected at rate of 98.4% in 2005 and 98.7% in 2008. A significant decrease in the triple dhfr (108Asn/51Ile/59Arg) mutation was observed from 2005 (78.6%) to 2008(56.4%) (p<0.01). The quadruple mutations of dhfr (108Asn/51Ile/59Arg)/dhps437Gly were significantly declined from 78.6% in 2005 to 53.8% in 2008 (p<0.01) while quintuple mutations (dhfr (108Asn/51Ile/59Arg)/dhps437Gly/dhps540Glu) showed a reduction from 60.6% to 37.2% after three years (p<0.01). In conclusion, the decline in the prevalence of dhfr/dhps combination mutations might indicate the re-emergence of sensitive parasites in the population following SP withdrawal. Therefore, further monitoring and assessment is important to determine the feasibility of re-introduction of SP alone or in combination as a more affordable and safer drug in the future in Ethiopia.

Molecular characterization of β-thalassemia in four communities in South Gujarat—codon 30 (G → A) a predominant mutation in the Kachhiya Patel community

Different thalassemia mutations have been reported in various ethnic groups and geographical regions in India. In this study, we have investigated Kachhiya Patel, Dhodia Patel, Modh Bania, and Muslim communities of Surat, Gujarat to identify molecular defects causing β-thalassemia in them. Covalent reverse dot blot hybridization technique was used to detect six common Indian β-thalassemia mutations while the seventh mutation (619-bp deletion) was identified by PCR. The less common mutations were detected by amplification refractory mutation and the uncharacterized samples were directly sequenced. Characterization of β-thalassemia mutations was carried out in a total of 175 unrelated β-thalassemia trait cases. We identified IVS 1 nt 5 (G → C) in 31 out of 65 Muslims, codon (Cd) 41/42 (-CTTT) in 14 out of 16 in Modh Banias, Cd 15 (G → A) in 19 out of 24 Dhodia Patels. The most significant observation was an uncommon mutation; Cd 30 (G → A) detected in 61 out of 70 Kachhiya Patels. The 619-bp deletion was detected in 6 out of 10 Muslim-Memons. Many other rare mutations like Cd 15 (-T), Cd 8 (-AA), -88 (C → A), Capsite +1 (A → C), Cd 16(-C), and Cd 5 (-CT) were detected. To our knowledge, our study is the first to characterize β-thalassemia mutations in the Kachhiya Patel community. This study will facilitate molecular analysis and prenatal diagnosis in these four communities.

Spectrum of α-thalassemia mutations including first observation of - -FIL deletion in Hatay Province, Turkey

Alpha thalassemia (α-thal) is one of the most common genetic disorders in the world. It is characterized by the absence or reduced expression of α-globin genes. The frequency of α-thal mutations in the province of Hatay in South Turkey is unknown. Therefore, in the present study, we aimed to investigate the spectrum of α-thal mutations in this province. Three hundred and nine patients were tested for α-thal mutations by using reverse dot blot hybridization technique and nine different mutations were detected in 97 of them. Among the 9 different mutations found, the most frequent mutations were the -α(3.7) (43.81%), -α2(-5nt) (6.70%), - -(MED) (5.67%) and α2(Poly A2) (2.57%). In the present study, - -(FIL) mutation was detected in a patient for the first time in Turkey. Our results indicated that α-thal mutations are highly heterogeneous and -α(3.7) is the most prevalent mutation in Hatay province of South Turkey. In addition, - -(FIL) mutation was detected in a patient for the first time in Turkey. This new finding may contribute to the establishment of a national mutation database and genetic counseling.