Conventional Restriction Fragment Length Polymorphism Research Articles

Profiling temporal dynamics of acetogenic communities in anaerobic digesters using next-generation sequencing and T-RFLP

Acetogens play a key role in anaerobic degradation of organic material and in maintaining biogas process efficiency. Profiling this community and its temporal changes can help evaluate process stability and function, especially under disturbance/stress conditions, and avoid complete process failure. The formyltetrahydrofolate synthetase (FTHFS) gene can be used as a marker for acetogenic community profiling in diverse environments. In this study, we developed a new high-throughput FTHFS gene sequencing method for acetogenic community profiling and compared it with conventional terminal restriction fragment length polymorphism of the FTHFS gene, 16S rRNA gene-based profiling of the whole bacterial community, and indirect analysis via 16S rRNA profiling of the FTHFS gene-harbouring community. Analyses and method comparisons were made using samples from two laboratory-scale biogas processes, one operated under stable control and one exposed to controlled overloading disturbance. Comparative analysis revealed satisfactory detection of the bacterial community and its changes for all methods, but with some differences in resolution and taxonomic identification. FTHFS gene sequencing was found to be the most suitable and reliable method to study acetogenic communities. These results pave the way for community profiling in various biogas processes and in other environments where the dynamics of acetogenic bacteria have not been well studied.

Epidemiological Investigations and Molecular Characterization of 'Candidatus Phytoplasma solani' in Grapevines, Weeds, Vectors and Putative Vectors in Western Sicily, (Southern Italy).

Bois noir is caused by ‘Candidatus Phytoplasma solani’, and it is one of the most important and widespread diseases in the Euro-Mediterranean region. There are complex interactions between phytoplasma and grapevines, weeds, and vectors. These ecological relationships can be tracked according to molecular epidemiology. The aims of the 2-year study (2014–2015) were to describe incidence and spatial distribution of Bois noir in a vineyard with three grapevine varieties in Sicily, and to identify the molecular types of the tuf and vmp1 genes in these naturally infected grapevines, according to the potential reservoir plants and vectors. Disease incidence in 2015 was significantly higher in ‘Chardonnay’ (up to 35%) than for ‘Nero d’Avola’ and ‘Pinot noir’ (<5%). All grapevine, weed, and insect samples were infected by ‘Ca. P. solani’ tuf-type b. Most of the collected insects were strictly related to Vitis spp. and belonged to Neoaliturus fenestratus, Empoasca spp., and Zygina rhamni. The characterization of the vmp1 gene revealed six different vmp types in grapevines (V1, V4, V9, V11, V12, V24), three in weeds (V4, V9, V11), and four in insects (V4, V9, V11, V24). Notably, V4, V9, appear both in hosts and vectors, with V9 predominant. Virtual restriction fragment length polymorphism (RFLP) analysis based on the nucleotide sequences supported the data of the conventional RFLP. Connections between the molecular data recorded in the vineyard ecosystems and the application of innovative tools based on the geostatistical analysis will contribute to further clarification of the specific ecological and epidemiological aspects of ‘Ca. P. solani’ in Sicily.

Molecular characterization of group 16SrI and 16SrIII phytoplasmas associated with loofah witches’ broom disease in Sinaloa, Mexico

Loofah [Luffa acutangula (L.) Roxb.] is grown in some regions of Asia, Africa and the Americas. This vegetable is well known for its high nutritional and medicinal value. Loofah plants showing typical symptoms of phytoplasma infection, such as witches’ broom, fruit deformation, flower proliferation and phyllody were observed in a commercial field in Sinaloa, Mexico, in January 2015. Nineteen symptomatic loofah plants and seven asymptomatic weeds (Helianthus annuus, Sarcostemma sp., Chenopodium sp., Amaranthus palmeri, Rumex sp., Ambrosia sp., and Raphanus sp.) growing nearby were collected and analyzed by phytoplasma-specific nested PCR. Phytoplasmas were detected in 79% of symptomatic loofah plants and in two of the seven weeds. Conventional and virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rRNA-encoding gene sequences indicated the presence of strains of the 16SrIII and 16SrI phytoplasma groups in loofah, which occurred as both single and mixed infections. The phylogenetic analysis confirmed four new phytoplasma strains associated with loofah diseases, two ‘Candidatus Phytoplasma asteris’-related strains (16SrI-AG and 16SrI-AH), and two ‘Candidatus Phytoplasma pruni’-related strains (16SrIII-AA, and 16SrIII-AB). Furthermore, phytoplasmas belonging to groups 16SrI and 16SrIII also were identified in the weeds H. annuus and A. palmeri, respectively; 16SrIII-AC represents a new phytoplasma subgroup described in A. palmeri in Sinaloa. To our knowledge, this is the first report of loofah disease associated with phytoplasmas belonging to the 16SrI and 16SrIII groups in Mexico.

Eleven genetic variants of seven important candidate genes involved in manifestation of type 2 diabetes mellitus

BackgroundDiabetes especially type 2 diabetes mellitus (T2DM) is securing the rank under five around the world and leading cause of death cardiac disease and cancer. T2DM is a severe inflammatory state mainly associated toobesityand enhanced oxidative stress. ObjectivesWe have investigated eleven genetic polymorphisms of seven important genes viz. IL-10, TNF-α, IL-6, adiponectin, IL-1, IL-18, and Vitamin D-receptor. We have focused to investigate the haplotypes, gene-gene interactions and their role in determining individual susceptibility to T2DM and their manifestation. MethodsEleven genetic variants of seven important candidate genes in 440 T2DM individuals and 440 controls were genotyped using conventional polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). All calculations were done by SPSS software and LD were analysed by haploview (SHEsis software). ResultsGenotypic frequency of rs1800872, rs1800795, rs2241766, rs17846866 and rs1946518 showed highly significant association in our population. While allele frequencies of rs1800795, rs2241766 and rs1946518 showed highly significant association. The allele set of “CGGAGGTAFTB”, “CGGAGTTAFTB” and “CGGATGTCFTB” increase the chance of diabetes up to 1.6 times. This study reflects that these individuals are more susceptible of having T2DM. ConclusionsThe present study will provide a new insight in the development and the manifestation of T2DM. These genetic studies showing gene interaction for the susceptibility of the disease may be used as prognostic markers and alter treatment strategies for T2DM.

Cytology and molecular study for GSTP1 effect on asthma Iraqi patients

BackgroundGST belongs to a super family of phase II detoxification enzyme and it plays an important role in preventing the damage that may occur due to reactive water-soluble compounds generated by the association of reactive intermediates with glutathione.MethodIn the present study, we analyzed the frequencies of GSTP1 polymorphism among the Iraqi population using PCR–RFLP technique. Fifty samples from bronchial asthma patients and fifty samples from control cases were subjected to conventional PCR and Restriction Fragment Length Polymorphism (RFLP) to detect GSTP1 genotype and measured different parameters together such as IgE, eosinophilic count, WBC, and so forth. Some of the cases were made to undergo sequence analysis and enrolled in NCBI GenBank with accession number (MG657249–MG657258). The GSTP1 polymorphism was determined using PCR and the resultant 176-bp fragment was subjected to RFLP and digested with BsamA1 to recognize the A–G transition at nucleotide.ResultsHomozygotes for Ile105 encoding allele resulted in 176-bp fragment found in 62% andVal105 encoding allele had two fragments of 91 and 85 bp in PCR was found in 4% of asthmatic patients. On the other hand, heterozygotes resulted in three fragments of 176, 91 and 85 bp seen in 34% of patients.ConclusionTo the best of the researcher’s knowledge, this is the first-of-its-kind report with regards to the role played by GSTP1 polymorphism in bronchial asthma among the Iraqi patients. Though the study outcomes do not support the large role played by GSTP1 gene polymorphism in the evolution of bronchial asthma disorder, future researchers are suggested to investigate more features for many promising results.

High-Throughput flaA Short Variable Region Sequencing to Assess Campylobacter Diversity in Fecal Samples From Birds.

Current approach to identify sources of human pathogens is largely dependent on the cultivation and isolation of target bacteria. For rapid pathogen source identification, culture-independent strain typing method is necessary. In this study, we designed new primer set that broadly covers flaA short variable region (SVR) of various Campylobacter species, and applied the flaA SVR sequencing method to examine the diversity of Campylobacter spp. in geese fecal samples (n = 16) with and without bacteria cultivation. Twenty-three Campylobacter strains isolated from the 16 geese fecal samples were grouped similarly by conventional flaA restriction fragment length polymorphism (RFLP) method and by the flaA SVR sequencing method, but higher discriminant power was observed in the flaA SVR sequencing approach. For culture-independent flaA SVR sequencing analysis, we developed and optimized the sequence data analysis pipeline to identify as many genotypes as possible, while minimizing the detection of genotypes generated by sequencing errors. By using this pipeline, 51,629 high-quality flaA sequence reads were clustered into 16 operational taxonomic units (=genotypes) by using 98% sequence similarity and >50 sequence duplicates. Almost all flaA genotypes obtained by culture-dependent method were also identified by culture-independent flaA SVR MiSeq sequencing method. In addition, more flaA genotypes were identified probably due to high throughput nature of the MiSeq sequencing. These results suggest that the flaA SVR sequencing could be used to analyze the diversity of Campylobacter spp. without bacteria isolation. This method is promising to rapidly identify potential sources of Campylobacter pathogens.

Association analysis between four vitamin D receptor gene polymorphisms and developmental dysplasia of the hip

Developmental dysplasia of the hip (DDH) is a congenital condition characterized by abnormality in acetabulum size and/or shape. The incidence rate of DDH differs between different populations with risk factors including positive family history, breech presentation, sex, firstborn status, side of the hip, mode of delivery and oligohydramnios. It is recognized that DDH has a genetic component that exhibit autosomal dominant patterns. Many candidate genes have been studied and found to be associated with the disease; most of them are normally involved in cartilage development and joint metabolism. In this study, the association of four single-nucleotide polymorphisms (SNPs) (rs731236, rs1544410, rs7975232 and rs2228570) in the vitamin D receptor (VDR) gene was studied by a case-control analysis. The study sample involves 50 cases with confirmed DDH presentation and 50 nonDDH controls. SNPs were genotyped using conventional polymerase chain reaction (PCR) and restriction fragment-length polymorphism (RFLP) techniques. Genotype and allele frequencies were analysed using SPSS software. No significant associations were found between the VDR polymorphisms analysed and DDH. Further work need to be performed using genomewide analysis to elucidate the genetic basis of DDH.

Development of a simple and practical method of discrimination between Vibrio furnissii and V. fluvialis based on single-nucleotide polymorphisms of 16S rRNA genes observed in V. furnissii but not in V. fluvialis

Vibrio furnissii and V. fluvialis are closely related, the discrimination of which by conventional biochemical assay remains a challenge. Investigation of the sequence of the 16S rRNA genes in a clinical isolate of V. furnissii by visual inspection of a sequencing electropherogram revealed two sites of single-nucleotide polymorphisms (SNPs; positions 460 A/G and 1261 A/G) in these genes. A test of 12 strains each of V. fluvialis and V. furnissii revealed these SNPs to be common in V. furnissii but not in V. fluvialis. Divergence of SNP frequency was observed among the strains of V. furnissii tested. Because the SNPs described in V. furnissii produce a difference in the target sequence of restriction enzymes, a combination of polymerase chain reaction (PCR) of the 16S rRNA genes using conventional primers and restriction fragment length polymorphism analysis using Eco RV and Eae I was shown to discriminate between V. fluvialis and V. furnissii. This method is simple and alleviates the need for expensive equipment or primer sets specific to these bacteria. Therefore, we believe that this method can be useful, alongside specific PCR and mass spectrometry, when there is a need to discriminate between V. fluvialis and V. furnissii.

Discrimination between Onchocerca volvulus and O. ochengi filarial larvae in Simulium damnosum (s.l.) and their distribution throughout central Ghana using a versatile high-resolution speciation assay.

BackgroundGenetic surveillance of the human filarial parasite, Onchocerca volvulus, from onchocerciasis endemic regions will ideally focus on genotyping individual infective larval stages collected from their intermediate host, Simuliid blackflies. However, blackflies also transmit other Onchocerca species, including the cattle parasite O. ochengi, which are difficult to distinguish from the human parasite based on morphological characteristics alone. This study describes a versatile approach to discriminate between O. volvulus and O. ochengi that is demonstrated using parasite infective larvae dissected from blackflies.ResultsA speciation assay was designed based on genetic differentiation between O. volvulus and O. ochengi mitochondrial genome sequences that can be performed in high-throughput high-resolution melt (HRM)- or lower throughput conventional restriction fragment length polymorphism (RFLP) analyses. This assay was validated on 185 Onchocerca larvae dissected from blackflies captured from 14 communities in Ghana throughout 2011−2013. The frequency of O. ochengi was approximately 67 % of all larvae analysed, which is significantly higher than previously reported in this region. Furthermore, the species distribution was not uniform throughout the study region, with 25 %, 47 % and 93 % of O. volvulus being found in the western-most (Black Volta, Tain and Tombe), the central (Pru) and eastern-most (Daka) river basins, respectively.ConclusionsThis tool provides a simple and cost-effective approach to determine the identity and distribution of two Onchocerca species, and will be valuable for future genetic studies that focus on parasites collected from blackflies. The results presented highlight the need to discriminate Onchocerca species in transmission studies, as the frequency of each species varied significantly between the communities studied.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1832-7) contains supplementary material, which is available to authorized users.

Appropriate Genotyping of Hepatitis B Virus in Iran.

Dear Editor We read, with great interest, the article by Karimi et al. (1) published in your journal recently, regarding the characterization of hepatitis B virus (HBV) genotypes in a group of patients with chronic hepatitis B infection. They found a prevalence of 73.9% and 26.1% for genotypes D and C, respectively, in Iranian patients. Bearing this context in mind, some points deserve to be considered. The patients were HBsAg-positive and a conventional polymerase chain reaction (PCR) methodology was used to detect DNA-positive samples. Subsequently, the authors applied genotype-specific real-time PCR to identify the genotypes. Only a fraction of the HBsAg-positive patients (19.82%) tested positive by conventional and real-time PCR. The use of standard PCR as a protocol for detecting DNA-positive samples was the major concern. Basically, all HBsAg-positive individuals should be tested positive by PCR, regardless of the type of PCR applied. The first weakness of the study was the sensitivity of PCR. In the field of clinical virology, nucleic acid amplification technology (NAT) is considered superior to conventional serology; therefore, samples testing positive by serological tests but negative by NAT is indicative of an error in the type of NAT used. Based on the article, it seems that the authors used multiplex real-time PCR to identify HBV genotypes; however, there is no data regarding the optimization of the method. Moreover, there is no information on the sequence of primers and probes used or any references that support the reliability of the kit. In such situations, real-time PCR can provide erroneous results, especially in the identification of minor variants. Therefore, the second weakness of the study was that the real-time PCR results were not confirmed by a specific approach such as sequencing. Previous and recent investigations carried out by the Iranian Hepatitis Network on the distribution of HBV among patients across different clinics have indicated that the main circulating genotype in Iran is D (2, 3). Some reports on the presence of genotypes apart from D in Iran have been published; however, they suffer from the same weaknesses as those mentioned above with respect to the methodology used. Previous surveys used fragment-based conventional PCR (4) and restriction fragment length polymorphism (RFLP) (5, 6). Although the sensitivity of RFLP is highly appreciated, RFLP and primer-specific methods are not very helpful in identifying minor variants in a quasispecies. Essentially, when a dominant variant exists in a population, the minor species might be ignored owing to their low proportion compared to that of the master sequence. Thus, sequencing is the gold standard method for the identification of genotypes. Owing to its adequate sensitivity, it is highly suitable for such purposes. Moreover, even the sensitivity of sequencing is not optimal for identifying all the possible variants. Therefore, the best option for accurate genotyping might be subjecting the amplicons to cloning plus sequencing. The Iranian hepatitis network suggests that all national labs and research centers use a standard (not necessarily unique) methodology for HBV genotypic characterization. A sensitive real-time PCR method could be used as an appropriate screening technique. Thus, regardless of the NAT type, we propose subjecting the amplified cloned extracted material to direct sequencing.

Year in review 2012: Lung cancer, respiratory infections, tuberculosis, pleural diseases, bronchoscopic intervention and imaging

This Year in Review series addresses the most relevant articles published in Respirology and other respiratory medicine journals during 2012 concerning five specific areas that we consider to be of importance to practicing pulmonologists, namely lung cancer, respiratory infections, tuberculosis (TB), pleural diseases, and interventional pulmonology and imaging. Some important findings that will be commented on more in depth are that: 1) screening for lung cancer using low-dose computed tomography (CT) in high risk populations is promising, although not firmly established, 2) an enhanced CURB (which stands for confusion, urea, respiratory rate, blood pressure) score as well as the Japanese A-DROP (age, dehydration, respiratory failure, orientation disturbance, and pressure) prognostic scale are as accurate as the pneumonia severity index (PSI) scoring system to predict mortality in patients with community-acquired pneumonia (CAP), 3) randomized trials are urgently needed to optimize multidrug-resistant (MDR)-TB treatment, 4) the use of video-assisted thoracoscopic surgery (VATS) to quantify pleural tumor burden and, if feasible, perform an intrathoracic cytoreduction in patients with malignant effusions secondary to ovarian cancer (OC) may have a significant impact on further patient management plans, and 5) respiratory endoscopy and its different diagnostic and therapeutic modalities, is a safe procedure with overall complication rates less than 1%.

Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay

Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.

Genetic variations in a well conserved 5′-untranslated region of hepatitis C virus genome isolated in Pakistan

The diversity and extent of sequence variations between hepatitis C virus (HCV) isolates from Pakistan were studied and the probable effects of these variations were assessed on secondary viral structures. Sequencing and phylogenetic analysis was performed on 33 samples, of which 25 were typed as genotype 3 by RFLP (restriction fragment length polymorphism) and 8 remained unresolved. Rooted neighbour-joining (NJ) tree revealed that 28 isolates were HCV type 3a and 5 isolates were typed as 3b. The majority of unresolved samples clustered in a different branch of genotype 3, supported by a bootstrap value of 71%. Another, cluster, cluster I, was found to have a bootstrap value of 81%. Genetic distance values showed significant diversity of isolates in these two clusters compared to the reference sequences. Pair-wise comparison showed the presence of additional restriction sites of HaeIII and RsaI in unresolved isolates. In conclusion, unique sequence variability was observed in the 5′-UTR of HCV type 3 isolates from Pakistan. One of the reasons for this sequence variability is the presence of mutations, which are additional restriction sites in the 5′-UTR. These mutations were also responsible for failure of conventional RFLP to type some of the HCV isolates.

Natural Coinfection with 2 Parvovirus Variants in Dog

To the Editor: Canine parvovirus (CPV) emerged in the late 1970s, presumably by mutations in feline panleukopenia virus, and became a major viral pathogen of dogs worldwide (1). Between 1979 and 1981, the original type 2 virus (CPV-2) was replaced by a new genetic and antigenic variant, type 2a (CPV-2a). Between 1983 and 1984, CPV-2a was replaced by type 2b (CPV-2b), which differs from type 2a by only 1 epitope located at residue 426 of the VP2 capsid protein (2). CPV-2 does not replicate in cats, but the new variants replicate in dogs and cats (3). Recently, an antigenic change has been observed in a new strain, CPV-2c, isolated from domestic dogs in Italy (4). This variant was also detected in Vietnam (5), other countries in Europe (6), and the United States (7). CPV-2c was recently detected in cats (8) and is characterized by a replacement of aspartic acid with glutamic acid at residue 426 of the VP2 capsid protein. To identify CPV types 2, 2a, and 2b, PCR methods were developed (9). However, these methods could not distinguish type 2c from type 2b (4). Consequently, we used a PCR–restriction fragment-length polymorphism (RFLP) assay with endonuclease MboII. This enzyme can distinguish type 2c from other CPVs (4). Recently, a real-time PCR assay based on minor groove binder (MGB) probe technology was developed for rapid identification and characterization of the antigenic variants. This assay is based on 1 nucleotide polymorphism in the VP2 gene (10). In June 2006, a 10-week-old female dog (PT-32/07) was brought to the veterinary clinic in Figueira da Foz, Portugal, with clinical signs of parvovirus infection, after an episode of gastrointestinal disease in her littermates. Three littermates, also brought to the clinic, showed no signs of infection. None of the dogs were vaccinated against CPV. Clinical signs in dog PT-32/07 were lethargy, anorexia, vomiting, diarrhea, and a temperature of 39.3°C. Identical signs were observed in 1 littermate 3 days later; the 2 other dogs did not show any signs other than lethargy and loose stools. Rectal swab samples from all dogs were screened for CPV by using an immunomigration rapid test (Synbiotics Corporation, Lyon, France). Two of the dogs showed negative results, and 2 showed positive results. Feces, serum, and lingual swab samples were positive for parvovirus DNA. DNA was quantified by using a real-time PCR with TaqMan technology performed in an i-Cycler iQ (Bio-Rad Laboratories, Milan, Italy). CPV variants were characterized by using MGB probe technology. This technology uses type-specific probes labeled with different fluorophores (FAM and VIC) that can detect single nucleotide polymorphisms between CPV types 2a/2b and 2b/2c (10). MGB probes specific for type 2b were labeled with FAM in both type 2a/2b and 2b/2c assays, and MGB probes specific for type 2a (type 2a/2b assay) and type 2c (type 2b/2c assay) were labeled with VIC. All specimens from 1 dog (PT-32/07) were positive for the 2 variants of CPV type 2 (CPV 2b and CPV 2c). Conversely, of the 3 littermates, 2 were positive for CPV type 2b and 1 was positive for CPV type 2c in all samples (Table). Table Detection by minor groove binder probe assay of CPV antigenic variants in different specimens from dogs from the same litter (10 weeks old), Portugal, 2006* A conventional PCR and RFLP analyses were performed by using the method of Buonavoglia et al. (4) with known positive CPV-2b and CPV-2c samples as controls to confirm our findings. The 583-bp PCR product obtained from the coinfected dog by using primer pair 555for/555rev was digested with MboII. Digestion generated 2 fragments (≈500 and 80 bp) in all dog samples. The CPV-2c control sample showed 2 fragments (≈500 and 80 bp), and CPV-2b control sample was not digested with MboII. We report CPV-2b and CPV-2c variants in samples from a dog with littermates that were positive for CPV-2b or CPV-2c during an episode of gastrointestinal disease. Coinfection with multiple CPV variants that showed high genetic diversity in the VP2 gene has recently been reported in a domestic cat (8). Continuous and rapid evolution of CPV may cause serious problems in diagnostic testing and vaccine efficacy. Antigenic variation may negatively affect vaccine efficacy if changes occur at major antigenic sites. Thus, continuous monitoring for novel genetic and antigenic virus types is needed. Additional studies are in progress to characterize nucleotide sequences of all CPV isolates from this case.

Improved Real-Time Multiplex Polymerase Chain Reaction Detection of Methylenetetrahydrofolate Reductase ( MTHFR) 677C>T and 1298A>C Polymorphisms Using Nearest Neighbor Model-Based Probe Design

The disorders of folate metabolism caused by methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms may lead to several disease states including coronary heart disease, venous thrombosis, and several types of cancer. We have developed a real-time multiplex single-tube polymerase chain reaction procedure on the LightCycler for the detection of the two most commonly occurring variants, 677C>T and 1298A>C, in the MTHFR gene. An improved probe design, based on the nearest neighbor model for nucleic acid-probe duplex stability, resulted in a better separation (DeltaTm approximately 10 degrees C) of melting peaks of the wild-type and mutant alleles than that by the existing method (DeltaTm approximately 3 degrees C) for specimens heterozygous for the 1298A>C polymorphism. Of the 333 blood specimens analyzed by this procedure, we did not find any samples that gave ambiguous results. The specimens with homozygous mutation for one polymorphism were of the wild type for the other variant. The assay was validated by the comparison of the genotyping results of 50 blood specimens from the LightCycler polymerase chain reaction with the conventional restriction fragment length polymorphism procedures. There was 100% concordance of the test results obtained by the two techniques. This assay is reliable, economical, and can be performed by less trained technologists compared with the procedure performed by the conventional restriction fragment length polymorphism technique.

Surface bacterial community, fatty acid profile, and antifouling activity of two congeneric sponges from Hong Kong and the Bahamas

Bacterial communities on the surfaces of 2 congeneric sponges, Mycale adhaerens from Hong Kong and M. laxissima from the Bahamas, were compared using conventional cultivation techniques and terminal restriction fragment length polymorphism (TRFLP) analysis — a culture- independent DNA fingerprinting technique. The bacterial community on the Hong Kong sponge was more diverse, in terms of the number and type of species isolated, and different from that on the Bahamas sponge, as evident from distinct clusters formed in TRFLP analysis. Distinctive bacterial types (i.e. TRFs) were commonly found on both sponge surfaces, but none of their bacterial isolates were common. At tissue level concentration, extract of the Hong Kong sponge inhibited the growth of a wide range of bacteria isolated from the Hong Kong reference surface, but did not affect any of those isolated from the Bahamas reference surface, indicating highly specific antibacterial activity. Extracts of both sponges, when incorporated into hydrogels and exposed to the natural environment for bacterial film development, dramatically altered the bacterial community in the films, either by shifting the bacterial composition or decreasing bacterial density. Settlement assays of the resulting films using larvae of the polychaete Hydroides elegans showed that the filmed hydrogels with Hong Kong sponge extracts either inhibited larval settlement or were toxic to the larvae; however, those with the Bahamas sponge extracts had no observable effect. HPLC (high performance liquid chro- matography) and GC-MS (gas chromatography-mass spectrometry) analyses revealed different chemical profiles in the extracts; the Hong Kong sponge had a more diverse fatty acid profile. Our results suggest that the 2 congeneric sponges from geographically separated regions have 'species- specific', surface-associated bacterial communities and antifouling activities, which might be due to the differences in the chemical and fatty acid compositions of the 2 sponges. Alternately, different sponge-associated bacterial communities may reflect habitat differences in sympatric bacterial and fouling communities.

Rapid and Accurate Genotyping of YMDD Motif Variants in the Hepatitis B Virus Genome by an Improved Reverse Dot Blot Method

By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.

A high throughput single-stranded conformation polymorphism assay for detection, subtyping and genotyping of influenza viruses

We have developed a high throughput single-stranded conformation polymorphism/capillary electrophoresis genotyping assay (SSCP/CE) to detect the presence of influenza virus and to determine its serotype, subtype and genotype. We configured the assay in two slightly different formats to suit the two applications: (i) To detect the presence of influenza virus and to determine its serotype and subtype in the clinical samples and (ii) to determine the genetic configuration of influenza virus reassortants generated by co-infection of two influenza virus strains in chicken embryo kidney (CEK) cells. We applied this newly developed assay to screen a large number of reassortants for a specific 6:2 genetic configuration characteristic of the FluMist Vaccines which exhibits two unique features: (i) the HA and NA segments from the circulating wild-type strains to confer immunogenicity and (ii) the internal segments of the master donor virus (MDV) to impart the attenuation phenotype. To evaluate the accuracy of the SSCP/CE assay, we analyzed 400 gene segments from approximately 50 different reassortant viruses and compared the SSCP/CE results with those obtained by the more conventional restriction fragment length polymorphism (RFLP) analysis. We found a very high concordance (98%) between the two sets of data.

Single step high-throughput determination of Toll-like receptor 4 polymorphisms

Background: Toll-like receptors are central components of host defence in humans, responsible for recognition of pathogen-associated molecular patterns and activation of innate immunity. Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS) and other microbial components, thereby initiating the expression and release of pro-inflammatory cytokines. The common, frequently co-segregating allelic variants Asp299Gly and Thr399Ile have been related to susceptibility to gram-negative infections and sepsis and may be involved in the development of atherosclerosis. Identification of TLR4 Asp299Gly and Thr399Ile genotypes can be important for examination of genotype/phenotype relationships as well as for individual risk assessment of patients. Methods: TLR4 Asp299Gly and Thr399Ile genotypes were detected by a single tube polymerase chain reaction (PCR), based on exonuclease degradation of dual labelled allele-specific oligonucleotides. The assay results were compared with conventional restriction fragment length polymorphism (RFLP) analysis. Results: Genotypes of 345 individuals were determined simultaneously in a single PCR assay. Allele frequencies for our population were 6.8% for the TLR4 Asp299Gly polymorphism and 6.4% for the Thr399Ile polymorphism. Validation by RFLP analysis revealed a correct detection of all genotypes. Conclusions: We have developed a novel method for the detection of the TLR4 Asp299Gly and Thr399Ile mutations, permitting rapid genotyping which should be useful for large-scale population studies as well as applicable for routine clinical testing.

Single nucleotide polymorphism genotyping by on-line liquid chromatography–mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI–MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 m M butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene–divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC–ESI–MS a potent tool for the routine characterization of SNPs in forensic science.