Restriction Fragment Length Polymorphism Results Research Articles

RHC genotyping in Chinese Han population

BackgroundThe Rh blood group system is characterized by its complexity and polymorphism, encompassing 56 different antigens. Accurately predicting the presence of the C antigen using genotyping methods has been challenging. The objective of this study was to evaluate the accuracy of various genotyping methods for predicting the Rh C and to identify a suitable method for the Chinese Han population.MethodsIn total, 317 donors, consisting 223 D+ (including 20 with the Del phenotype) and 94 D− were randomly selected. For RHC genotyping, 48C and 109bp insertion were detected on the Real-time PCR platform and −292 substitution was analyzed via restriction fragment length polymorphism (RFLP). Moreover, the promoter region of the RHCE gene was sequenced to search for other nucleotide substitutions between RHC and RHc. Agreement between prediction methods was evaluated using the Kappa statistic, and comparisons between methods were conducted via the χ2 test.ResultsThe analysis revealed that the 48C allele, 109bp insertion, a specific pattern observed in RFLP results, and wild-type alleles of seven single nucleotide polymorphisms (SNPs) were in strong agreement with the Rh C, with Kappa coefficients exceeding 0.8. However, there were instances of false positives or false negatives (0.6% false negative rate for 109bp insertion and 5.4-8.2% false positive rates for other methods). The 109bp insertion method exhibited the highest accuracy in predicting the Rh C, at 99.4%, compared to other methods (P values≤0.001). Although no statistical differences were found among other methods for predicting Rh C (P values>0.05), the accuracies in descending order were 48C (94.6%) > rs586178 (92.7%) > rs4649082, rs2375313, rs2281179, rs2072933, rs2072932, and RFLP (92.4%) > rs2072931 (91.8%).ConclusionsNone of the methods examined can independently and accurately predict the Rh C. However, the 109bp insertion test demonstrated the highest accuracy for predicting the Rh C in the Chinese Han population. Utilizing the 109bp insertion test in combination with other methods may enhance the accuracy of Rh C prediction.

Frequency of Transcription Factor 7-like 2 rs12255372 Mutation among Bangladeshi Adult Type 2 Diabetes Patients

ABSTRACT Background: While the etiology of type 2 diabetes mellitus (T2DM) is complex, genetic markers for T2DM are surfacing fast, and amidst such markers, the transcription factor 7-like 2 (TCF7L2) gene has been emerged as the most promising T2DM-causing gene. The intronic variant rs12255372 of the TCF7L2 gene has been found to be strongly associated with T2DM in different ethnic populations. Objectives: The objectives of this study were to determine the frequency of rs12255372 polymorphism in the TCF7L2 gene in Bangladeshi adult population with T2DM. Materials and Methods: This cross-sectional study was conducted in the Department of Endocrinology, Bangabandhu Sheikh Mujib Medical University. Eighty T2DM patients were recruited consecutively by nonrandom sampling, who signed the informed consent, history, and clinical features including height, weight, and waist circumferences were recorded. Blood samples were collected. Genomic DNA was extracted and single-nucleotide polymorphisms rs12255372 (G>T) were determined by the restriction fragment length polymorphism (RFLP) method. Statistical Analysis: RFLP results were analyzed by the gel electrophoresis to get the frequency of rs12255372 polymorphism. The independent segregation of alleles was tested for Hardy–Weinberg equilibrium using the Chi-square test of goodness of fit. P ≤0.05 was considered statistically significant. Results: The minor (T) allele frequencies (MAF) for rs12255372 (G>T) among T2DM was 28%. The observed genotype frequencies of rs12255372 (G>T) were different than those expected from the Hardy–Weinberg equation. Conclusion: The minor T allele frequency of rs12255372 (G>T) among T2DM participants was about one-fourth.

The Cyp2b6 Gene Polymorphism and Phenotypic Correlation of Efavirenz-Based Combination Therapy Among the Niger Delta Ethnic Population: Implications in Modern Pharmacogenomics.

PurposeDNA polymorphism describes the difference between individuals, groups, or ethnicities, races, etc., in terms of their DNA sequences or phenotypes as relates to drug metabolism. Using predictive genotyping of drug-metabolizing genes, we can develop individuals’ drug therapies that are less toxic and more effective. The main aim of the study was to evaluate genotype–phenotype-based correlation and incidence of genetic polymorphism of efavirenz blood levels among HIV/AIDS patients of the Niger Delta population.MethodsA study questionnaire was designed to obtained patients’ data, blood samples were obtained, plasma was separated from the serum using a centrifuge for 5 minutes at 4000 rpm for HPLC analysis, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis was conducted using Bsrl endonuclease enzyme to digest the PCR amplicons. Standard efavirenz was used at 0.5, 1, 2, 4, 16 mg/L to construct a calibration curve. Data were analyzed with SPSS software using chi-square test at p-value ≤0.5 and Microsoft excel 2013, while PCR and RFLP results were obtained after 1% Agarose gel electrophoresis, respectively.ResultsPhenotypic results showed that the participants had different efavirenz plasma concentrations. Six subjects (12%) had efavirenz plasma levels below 0.10 mg/L, considered ultra-rapid metabolizers (UMs), 22 (44%) 0.10 mg/L to 0.90 mg/L, classified as extensive metabolizers (EMs), 19 (38%) had 1.0 to 3.9 mg/L and were noted as intermediate metabolizers (IM), while 3 (6%) subjects showed efavirenz plasma levels from 4.0 mg/L to 6.0 mg/L, categorized as poor metabolizers (PM). RFLP results showed more than half of the population (56%) with a homozygous wild-type gene with CYP2B6*1*1 allele, 38% were CYP2B6*1*6 (heterozygous mutant) allele and 6% had homozygous mutant gene (CYP2B6*6*6 allele). Out of the 15 male subjects among the 50 patients that participated in the study, 8% were UM, 12% EM, 14% IM while no PM was observed, on the contrary, out of the 35 females participated in the study, 4% were observed as UM, 32% EM, 24% IM, while 6% were PM.ConclusionThere was no significant difference (p ≤ 0.05) between genotype and phenotype data for CYP2B6 polymorphism, among the HIV/AIDS patients that participated in this study. Genetic polymorphism of the CYP2B6 gene is prevalent among HIV/AIDs patients in the Niger Delta ethnic population on efavirenz-based HAART treatment, as the population having homozygous mutant gene or PM are >1% (6%).

The profile of inflammatory factors in dairy calves with Cryptosporidium infection.

The present study was conducted to investigate the detection and identification of Cryptosporidium species via molecular techniques and evaluate the serum concentrations of inflammatory factors in Cryptosporidium species. The fecal samples (n = 256) were collected from pre-weaned (≤ 2.00 months) calves and the positive samples were identified utilizing Ziehl-Neelsen staining. Nested species-specific multiplex PCR (nssm-PCR) and restriction fragment length polymorphism (RFLP) were used to identify the species and sub-species. The serum concentrations of IL-1β, IL-6, IL-12, TNF-α, and IFN-γ were also assessed. The results revealed that 10.54% of samples were positive. The results of Nested-PCR showed that 92.59% of the samples were positive for C. parvum while 7.41% were positive for C. andersoni. The results of RFLP confirmed 92.59% of the samples for C. parvum, 3.70% for C. muris / C. andersoni, and 3.70% for C. muris. The serum concentrations of IL-1β, IL-6, IL-12, TNF-α, and IFN-γ were significantly higher in the infected calves compared to those in healthy calves. However, the serum concentration of IFN-γ was significantly higher in the calves infected with C. parvum while the serum concentrations of TNF-α and IL-6 were significantly higher in those infected with C. andersoni . In conclusion, C. parvum was prevalent in the region and the calves demonstrated inflammatory responses to Cryptosporidium species.

Distribution characteristics and diversities of cbb and coxL genes in paddy soil profiles from southern China

The Calvin-Benson-Bassham cycle and Wood-Ljungdahl pathway are two well-known ones in the six carbon sequestration pathways, but the current knowledge of their occurrence in different layers of agricultural soil profiles is poor. In this study, the diversities of three genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), i.e., genes encoding the green-like (cbbLG) and red-like (cbbLR) forms of RubisCO I and encoding RubisCO II (cbbM), and the gene encoding carbon monoxide dehydrogenase large subunit (coxL) from five paddy soils in southern China were investigated by real-time quantitative polymerase chain reaction, restriction fragment length polymorphism (RFLP) analysis, and clone library. The abundances of the four genes rang ed from 107 to 109 copies g–1 soil, and the cbbLR gene outnumbered the other three genes in all soil samples, suggesting important roles they play in carbon dioxide (CO2) fixation. In addition, it was found that the copy numbers of cbbLR and cbbLG decreased with increasing soil depth, while the copy numbers of cbbM and coxL decreased in the shallow depths but increased with increasing soil depth. The results of RFLP showed a larger Shannon index (H) in the deeper soil layers among the four gene clone libraries, indicating that the community diversity in these soil layers was greater. The cbbLG gene had relatively low diversity (at genus level), and most of the sequences were classified as Sideroxydans and Thiobacillus. In contrast, the highly diverse groups were found in the other three gene clone libraries (cbbLR, cbbM, and coxL), most of which were distantly related to known sequences, even forming separate clusters. In summary, this study provides a new insight into CO2 fixers along agricultural soil profiles by comparing four bacterial genes.

Identification of ginseng radix in Chinese medicine preparations by nested PCR-DNA sequencing method and nested PCR-restriction fragment length polymorphism

Ginseng Radix, the dried root of Panax ginseng, is easily confused with other herbs of the Panax species by appearance. In Chinese medicine preparations, it is difficult to identify misuse of Ginseng Radix. In this study, the nested PCR and DNA sequencing methods were established for the identification of Ginseng Radix, whereas the combination of nested PCR and restriction fragment length polymorphism (RFLP) could differentiate Ginseng Radix from other Panax species and thus certify the use of impure raw material of Ginseng Radix in Chinese medicine preparations. Nested PCR and DNA sequencing methods were applied to verify ginseng ingredients in the preparations. Of the 58 samples analyzed, 48 samples contained P. ginseng, 8 P. quinguefolius, and 2 P. notoginseng. Utilizing restriction enzyme BstYI and DdeI, nested PCR products of the samples were digested for restriction fragment lengths of polymorphism (RFLP). It was proven that 23 of the 58 samples contained a mixture of Ginseng Radix and Panacis quinquefolii Radix. Twenty-five samples contained pure raw material of Ginseng Radix. Ginseng components of 10 samples were confirmed using a substitute of P. quinguefolius or P. notoginseng. The RFLP results were consistent with those of the sequencing method results.

Association analysis of ApoE gene polymorphisms among Egyptian patients with Alzheimer's disease

BackgroundAlzheimer disease (AD) is one of the leading causes of dementia among elderly. It is a progressive brain disorder associated with unusual behaviors, personality changes, and irreversible decline in thinking ability. Epsilon 4 allele of apolipoprotein E (ApoE) is considered as a major risk factor for AD in several populations. AimWe investigated APOE polymorphisms in a group of Egyptian patients with Alzheimer's disease. Subjects and methodsWe analyzed APOE polymorphisms in 53 Alzheimer patients who were 60 years or older and the study included a control group of 100 individuals. For APOE genotyping, we used conventional PCRs followed by Restriction Fragment Length Polymorphism (RFLP) analysis. Additionally, we performed Real time PCR analysis to confirm the results of RFLP and apply a more accurate method that is fast, simple, and cost-effective. ResultsWe observed that E4 allele was associated with 28.3% of Alzheimer patients as E3/E4 genotype (22.6%) and E4/E4 (17.0%), while we detected it in only 4% of the control group as E3/E4 genotype (8%). E3 allele has been shown in 69.8% of Alzheimer patients and in 93.5% of the control subjects. We have detected E2 allele in 18.9% of the cases and in 25% of the control group. RT-PCR analysis showed the exact patterns of genotype as determined by RFLP. ConclusionThere is a significant association between ApoE4 isomer and Alzheimer dementia in the Egyptian patients.We recommend conducting further studies to screen larger number of patients and confirm that ApoE4 could be considered as a reliable molecular marker for late onset Alzheimer disease (LOAD) among Egyptian elderly.

Possible association between -954G/C iNOS polymorphism in nasal polyposis. A case-control study in a population group of Northern Romania

Abstract BACKGROUND. Polymorphisms for genes encoding chemosensitive signalling proteins like NOS2 might contribute to the variability in individual susceptibility to nasal polyposis. NO produced by the inducible NO synthase enzyme NOS2A is generated at high levels in certain types of inflammation, so that the role of NOS2 might also be important in nasal polyposis etiopathogeny. MATERIAL AND METHODS. This is a cross-sectional, randomized, case-control study for the evaluation of the frequency of -954G/C NOS2A2 alleles among patients with nasal polyposis. The study included 91 cases of nasal polyposis diagnosed patients (nasal endoscopy and CT scan examination), and 117 healthy unrelated controls. NOS2 genotyping was carried out using PCR amplification of relevant gene fragment and it was followed by restriction enzyme digestion. Detection of the variant alleles was determined through analysis of resulting restriction fragment length polymorphism (RFLP) followed by gel electrophoresis. RESULTS. Molecular analysis revealed an increased frequency of NOS2 variant allele in the study group compared to the control group (p=0.019, OR=1.991, CI=1.08-3.67). A statistically significant finding was highlighted among allergic and nonallergic patients with nasal polyposis (p=0.046, OR=0.449. CI=0.208-0.969) and a relationship between nasal polyposis patients with asthma and non-asthmatic patients (p=0.119, OR=1.825, CI=0.875-3.80). CONCLUSION. The main finding of our study is that -954G/C polymorphism of NOS gene seems to be associated with an increased risk for nasal polyposis.

Molecular typing of Brucella species by PCR-RFLP and SSCP

PCR-RFLP and PCR-SSCP help to understand variabilities in Brucella spp. genome which in turn assist in planning epidemiological strategies for control of brucellosis in animal population and thereby human transmission. Restriction fragment length polymorphism (RFLP) analysis was carried out for BCSP31 (223 bp) gene of 15 isolates using restriction enzyme HaeII and BsaBI. All isolates yielded a similar restriction pattern as that of reference strains i.e. 189 + 34 bp and 164 + 60 bp fragments with HaeII and BsaBI restriction enzyme, respectively. PCR- SSCP analysis of BCSP 31 gene of 33 Brucella spp. isolates was carried out. Seven different SSCP band patterns designated from “A” to “F” were observed. Out of 33 isolates of Brucella spp., 19 isolates showed SSCP profile similar to ATCC reference strains of Brucella spp. designated as band pattern “A” indicating no polymorphism. Whereas, 14 isolates showed polymorphism in SSCP band pattern designated from “B” to “G”. Band pattern “A” was the most common in 19 (57.58 %) isolates followed by “B” in 6 (18.18 %) isolates and “G” in 4 (12.12 %) isolates, whereas “C”,”D”,”E”, and “F” band patterns were found in each of 1 (3.03 %) isolate. SSCP band pattern “A” was observed most commonly in buffalo (10) followed by human (6) and cattle (3). The SSCP band pattern “B” was found in equal proportion in cattle and buffalo (3 each). The “C” and “E” SSCP band patterns were observed in cattle, whereas “D” and “F” SSCP band pattern was noticed in buffalo and human isolates, respectively. The “G” SSCP band pattern was observed in 3 cattle and 1 goat isolate. In cattle-ABCEG, buffalo-ABD, goat-G and in human-AF band patterns were seen. Comparative results of RFLP and SSCP in 15 isolates showed that, PCR-SSCP is more sensitive than RFLP for detection of polymorphism in BCSP31 gene.

Comparative Study of Campylobacter spp. Isolated from Children With Gastroenteritis in Bahonar Hospital, Karaj, Using PCR and RFLP

Background: Campylobacter species are responsible for the majority of cases of food-borne gastroenteritis. The sources of the disease outbreaks are often contaminated water or milk, and consumption of undercooked poultry product is the main cause of sporadic campylobacteriosis cases. Objectives: The aims of this study were to determine the prevalence of Campylobacter gastroenteritis in children and to differentiate the interfering species using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods at the Bahonar hospital in Karaj, Iran. Patients and Methods: A total of 150 stool samples were collected from children under 10 years old during the summer of 2014. PCR was performed using genus- and species-specific primers and RFLP was done using AluI and TasI enzymes. Results: The results showed the amplification of 400 and 491 bp segments and Campylobacter contamination in 30 (20%) samples; 5 out of 30 Campylobacter positive samples (16.66%) were identified as C. jejuni, 20 (66.66%) as C. coli, 3 (10%) as C. jejuni and C. coli (mixed infection), and 2 (6.66%) were identified as non-jejuni, non-coli Campylobacter using the PCR method. Following the evaluation of RFLP results, 7 positive samples (23.33%) showed the electrophoretic pattern of C. jejuni, 21 (70%) showed the electrophoretic pattern of C. coli, and 2 (6.6%) showed both of the patterns and mixed contamination with jejuni and coli species. The results of digestion with TasI did not show any C. lari or C. upsaliensis patterns. Conclusions: The results of this study showed high percentage of Campylobacter contamination in the tested stool samples. The other surprising finding was the high rate of Campylobacter coli positive samples; the difference between the results of PCR using species-specific primers (hipo and asp) and the RFLP method (electrophoretic patterns) in some of the positive samples confirms the hypothesis of variations in nucleotide sequences of the hipo primer binding site in some Iranian isolates of Campylobacters.

Mitochondrial ND1 gene mutation analysis in type II diabetes of Karaikudi population

Mitochondrial single nucleotide polymorphisms (SNPs) are significantly correlated with the development of diabetes mellitus . However, these polymorphisms are emerging to be considerably diverse among different population and regions. Sivaganga district is recently being given importance for genetic studies due to its rigid family structure. Hence, the present study is planned to investigate the correlation between mitochondrial SNP of ND1 gene with type II diabetes (T2D) of Karaikudi population of Sivaganga district. For this study, 206 samples including 105 T2D and 101 healthy volunteers were collected and screened for ND1 gene mutation. Genotyping for the ND1 gene was characterized by polymerase chain reaction, Restriction fragment length polymorphism (RFLP) followed by sequencing. In addition, clinical and biochemical variables were analyzed. RFLP and sequencing results showed G>A transition at 3316 position of ND1 gene in both T2D (6.67 %) and in control subjects (1.9 %). Since the frequency of the mutation was found high in T2D patients, we screened for the mutation in their family members and identified its maternal mode of inheritance. The result warrants for the detailed study to identify population based mitochondrial mutation association with disease development.

First Report of a New Subgroup 16Sr II-M 'Candidatus Phytoplasma aurantifolia' Associated with Witches'-Broom Disease of Tephrosia purpurea in India.

Wild indigo (Tephrosia purpurea (L.) Pers.) grows as a common weed throughout the Indian subcontinent. The plant has pinnate leaves, white or purplish flowers, and flat hairy pods, and is cultivated as a green manure crop. The plant extracts contain compounds such as tephrosin, an aromatic ester, prenylated flavonoid, and sesquiterpene (2) that have medicinal properties. The newly recognized disease, Tephrosia purpurea witches' broom (TPWB), was characterized by chlorosis, stunting, and proliferative branching, which were suggestive of phytoplasma infection during a field survey conducted in November 2013. To determine the presence of phytoplasma, 2 g of compound leaves from three symptomatic and asymptomatic plants were used for total DNA extraction using the CTAB method. The phytoplasma 16S rRNA gene was detected in all three symptomatic plants using nested PCR with universal phytoplasma primer pairs, P1/P7 followed by R16F2n/R16R2 (4). No amplification was observed in DNA isolated from asymptomatic plants. PCR fragments (1,246 bp in length) generated from symptomatic T. purpurea plants were sequenced directly using five different primers viz. 343R, 536F, 704F, 907R, and 1103F. TPWB phytoplasma 16S rRNA gene sequence (GenBank Accession No. HG792252) showed 99.12% homology with a 'Candidatus Phytoplasma aurantifolia' strain WBDL (U15442) when compared using the EzTaxon 16S rRNA database (3). Virtual restriction fragment length polymorphism (RFLP) analysis was carried out on the obtained sequence using iPhyClassifier (5). The virtual RFLP pattern derived from the HG792252 sequence was different to the reference patterns of previously established 16Sr groups and subgroups. The reference pattern of the 16Sr group II, subgroup C (AJ293216) was most similar with a similarity coefficient of 0.92, which placed it in a new subgroup, 16Sr II-M (1). Furthermore, virtual RFLP results were confirmed by digesting R16F2n/R16R2 amplicon with BstUI, DraI, HinfI, HpaI, and MseI restriction enzymes according to manufacturer's instructions. To our knowledge, this is the first report of a 'Ca. P. aurantifolia'-related strain associated with witches'-broom disease of T. purpurea in India.

Novel Phl-producing genotypes of finger millet rhizosphere associated pseudomonads and assessment of their functional and genetic diversity.

Genetic diversity of phlD gene, an essential gene in the biosynthesis of 2,4-diacetylphloroglucinol, was studied by restriction fragment length polymorphism (RFLP) in 20 Phl-producing pseudomonads isolated from finger millet rhizosphere. RFLP analysis of phlD gene displayed three patterns with HaeIII and TaqI enzymes. phlD gene sequence closely correlated with RFLP results and revealed the existence of three new genotypes G, H and I. Further, the phylogenetic and concatenated sequence analysis of the 16S rRNA, rpoB, gyrB, rpoD genes supported the hypothesis that these genotypes G, H and I were different from reported genotypes A-F. In all phylogenetic studies, the genotype G formed a distant clade from the groups of Pseudomonas putida and P. aeruginosa (sensu strictu), but the groups H and I were closely related to P. aeruginosa/P. stutzeri group. The Phl-producing pseudomonads exhibited antagonistic activity against Pyricularia grisea (TN508), Gaeumannomyces graminis (DSM1463), Fusarium oxysporum (DSM62297), Xanthomonas campestris (DSM3586) and Erwinia persicina (HMGU155). In addition, these strains exhibited various plant growth-promoting traits. In conclusion, this study displays the existence of novel Phl-producing pseudomonads genotypes G, H and I from finger millet rhizosphere, which formed taxonomically outward phylogenetic lineage from the groups of P. putida and P. aeruginosa (sensu strictu).

First Report of Two Distinct Phytoplasma Species, 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma asteris,' Simultaneously Associated with Yellow Decline of Wodyetia bifurcata (Foxtail Palm) in Malaysia.

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV 'Candidatus Phytoplasma cynodontis' group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the 'Candidatus Phytoplasma asteris' 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia.

First report of a ‘Candidatus Phytoplasma ulmi’ isolate affecting sapodilla trees in western Cuba

The cultivation of sapodilla (Manilkara zapota) has increased in the last five years in western Cuba as part of a fruit diversification programme to support the tourism industry, and as a potential export crop. Symptoms of witches' broom have recently been observed in sapodilla trees in a fruit farm located at Ceiba, in Artemisa western province (Fig. 1A) strongly contrasting with those healthy-looking sapodilla trees (Fig. 1B). Similar symptoms were previously associated with a phytoplasma in eastern Cuba (Acosta et al., 1). Therefore sapodilla plants from Ceiba were surveyed and tested for phytoplasmas. Total DNA was extracted from 0.5 g of leaf midribs of five symptom-bearing and five symptomless sapodilla plants collected (Murray & Thompson, 5), and used as a template for a nested PCR assay. Universal primer pairs that target the phytoplasma 16S rRNA gene, P1 (Deng & Hiruki, 3) and P7 (Schneider et al., 6) were used for the first reaction, and R16F2n/R16R2 (Gundersen & Lee, 4) for the nested reaction. Nested PCR products of expected size (approximately 1250 bp) were obtained from five symptom-bearing plants. PCR products were purified (Wizard SV Gel and PCR Clean-Up System, Promega, Madison, WI, USA) and cloned (pGEMT-Easy Vector, Promega). One individual clone per infected plant was sequenced using Macrogene Inc. Sequencing Service (Korea). The R16F2n/R16R2 sequences were trimmed, assembled into a consensuses using the Sdaten package (Bonfield & Whitwham, 2) and compared with reference sequences from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov). Sequences were subjected to in silico restriction fragment length polymorphism (RFLP) analysis with endonucleases AluI, BfaI, BstUI, HaeIII, HhaI, HinfI, HpaII, MseI and Tsp509 (pDRAW32 AcaClone Software), and then subjected to phylogenetic analysis with the Mega version 5.0 software (USA). The R16F2n/R16R2 sequences of the phytoplasma detected in the symptom-bearing sapodilla plants were 100% identical to each other. The consensus sequence (1171 nt) of the sapodilla phytoplasma (GenBank Accession No. KF500549) showed the highest sequence identity (98.04%) with the elm yellows (EY) phytoplasma strains from the 16SrV group ‘Candidatus Phytoplasma ulmi’, including the strain EY1 (AY197655). Phylogenetic analysis confirmed both sequence and RFLP results (Fig. 2) since the sapodilla phytoplasma clustered within the 16SrV phylogenetic branch closely related to the EY1 strain (AY197655). All RFLP profiles of the sapodilla phytoplasma were identical to those of the 16SrV group used as reference, except for the AluI, MseI and BstUI RFLP profiles. This suggests that the phytoplasma detected in sapodilla trees of western Cuba may be a member of a new subgroup. A phytoplasma of group 16SrII ‘Ca. Phytoplasma aurantifolia’ was previously identified in sapodilla plants of eastern Cuba showing witches' broom and leaf yellowing symptoms (Acosta et al., 1). However, this is the first record of a ‘Ca. Phytoplasma ulmi’ isolate identified in western Cuba affecting sapodilla plants. We thank Dr. Chrystel Olivier for her essential contributions to this work.

Association of polymorphisms in microRNA-binding sites and colorectal cancer in an Iranian population

MicroRNAs (miRNAs) are agents of post-transcriptional gene expression, and they can affect many functions of an individual cell or tissue from extracellular matrix production to inflammatory processes and tumor development. We aimed to determine the possible role of miRNA-binding site polymorphisms located in five cancer-related genes: IL-16, CDKN2A (p16), RAF1, PTGER4, and ITGB4 in colorectal cancer (CRC) risk modification in an Iranian population. This study was performed on 643 individuals (249 CRC cases and 394 healthy controls). We selected five cancer-related genes (IL-16, CDKN2A (p16), RAF1, PTGER4, and ITGB4) and investigated the genotypes of the 3' untranslated region miRNA-binding site polymorphisms in these genes in our study population. The restriction fragment length polymorphism results were confirmed by a direct sequencing method. We found a statistically significant difference between the rs1131445 polymorphism of the IL-16 gene and CRC. The frequencies of the genotypes TT, CT, and CC in controls were 51%, 40.4%, and 8.6%, respectively, and in cases were 41.4%, 44.1%, and 14.5%, respectively, which shows a significant association between the CC genotype of the rs1131445 polymorphism and CRC (P = 0.004). The frequency of the C allele in the CRC group was higher than in the controls, and the C allele of the rs1131445 polymorphism was found to be in association with CRC (P = 0.009). These associations remained significant after Bonferroni's correction for multiple testing. We found that the AA genotype of the rs743554 polymorphism in the ITGB4 gene and the T allele of the rs1051208 polymorphism of the RAF1 gene were associated with the risk of CRC in females; however, after Bonferroni's correction we found that they were non-significant. Finally, we can conclude that a significant relationship exists between the miRNA-binding site polymorphism of the IL-16 gene and CRC risk in the Iranian population.

P3.06 Comparison of High Resolution Melting and Restriction Fragment Length Polymorphism to Genotype Snp in Formalin Fixed Paraffin-Embedded Breast Cancer Tissue

ABSTRACT SNPs of NQO1, p53, and MDM2 have been correlated with pathological response or overall survival of breast cancer patients treated with anthracycline based chemotherapy. Therefore, the ability to detect these SNPs may be useful for treatment decision. In this study, we genotyped FFPE breast cancer samples to detect those SNP. Objective The aim of this study was to know the accuracy of HRM (High Resolution Melting) method to genotype SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G's in comparison to RFLP (Restriction Fragment Length Polymorphism) from archived formalin fixed paraffin embedded (FFPE) breast cancer blocks. Materials and Methods FFPE blocks were collected from anatomic pathology laboratory. DNA were isolated, and PCR amplified followed by either RFLP or HRM methods. Restriction enzymes HinfI, BstUI, and MspA1I were used in RFLP to genotype NQO1, p53, and MDM2 SNPs, respectively. The primer design which been used was the same between HRM and RFLP. The results of HRM were analyzed using melting, normalization, and differentiation plot curves. Sequencing technique has been used to resolve discrepancy between RFLP and HRM results. Results We screened SNPs on breast cancer FFPE samples using HRM and RFLP. The rates of genotyping agreement between both methods are 69, 23% (9/13 samples), 80% (8/10 samples), and 100% (7/7 samples) for NQO1, p53, and MDM2 SNPs, respectively. Conclusion HRM is a faster method than RFLP to genotype important breast cancer SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G. Nevertheless, RFLP is more reliable method than HRM when the used primers sets are not optimized for HRM.

First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman.

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman.

STAT4 polymorphism in a Chinese Han population with Vogt–Koyanagi–Harada syndrome and Behçet's disease

This study investigated the association of rs7574865 polymorphism in STAT4 with Vogt–Koyanagi-Harada (VKH) syndrome and Behçet's disease (BD) in a Chinese Han population. Genotyping of rs7574865 polymorphism in the STAT4 gene was performed using polymerase chain reaction restriction fragment length polymorphisms in 379 VKH patients, 366 BD patients, and 414 controls. Of the samples, 20% were sequenced to validate polymerase chain reaction restriction fragment length polymorphism results. A binary logistic regression analysis was used to assess the influence of the gender on the association of STAT4 polymorphism with BD. A significantly increased frequency of TT genotype of the STAT4 rs7574865 was observed in VKH patients ( p = 0.013). GT genotypic frequency was significantly lower in BD patients than in controls ( p = 0.003) However the significance of rs7574865 was lost in all tested BD patients when adjusted for gender ( p = 0.775). A significantly lower frequency of GT genotype and a significantly higher frequency of GG genotype was found in male BD patients compared with male controls ( p = 0.000458 and p = 0.009, respectively). Stratification analysis according to tinnitus, alopecia, poliosis, headache, and vitiligo for VKH syndrome and oral ulceration, genital ulceration, skin lesions and arthritis for BD failed to find any association between the tested single nucleotide polymorphism and any of the extraocular findings. Our results suggest that TT genotype of rs7574865 may be a susceptible factor for VKH syndrome in a Chinese Han population, and that GG genotype of this SNP may confer susceptibility in male BD patients.

Prospective Universal Application of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping To CharacterizeMycobacterium tuberculosisIsolates for Fast Identification of Clustered and Orphan Cases

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.