Multi-wall carbon nanotubes (MWCNTs) were modified to design a new DNA biosensor. Functionalized MWCNTs were equipped with gold nanoparticles (GNPs) (~15nm) (GNP-MWCNTCOOH) to construct DNA biosensors based on carbon-paste screen-printed (SPE) electrodes. GNP attachment onto functionalized MWCNTs was carried out by microwave irradiation and was confirmed by spectroscopic studies and surface analysis. DNA biosensors based on differential pulse voltammetry (DPV) were constructed by immobilizing thiolated single-stranded DNA probes onto GNP-MWCNTCOOH. Ruthenium (III) chloride hexaammoniate [Ru(NH3)6,2Cl(-)] (RuHex) was used as hybridization redox indicator. RuHex and MWCNT interaction was low in compared to other organic redox hybridization indicators. The linear response range for DNA determination was 1×10(-21) to 1×10(-9)M with a lower detection limit of 1.55×10(-21)M. Thus, the attachment of GNPs onto functionalized MWCNTs yielded sensitive DNA biosensor with low detection limit and stability more than 30days. Constructed electrode was used to determine gender of arowana fish.
A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.
A fluorometric assay is described for highly sensitive quantification of Escherichia coli O157:H7. Reporter oligos were immobilized on graphene quantum dots (GQDs), and quencher oligos were immobilized on gold nanoparticles (AuNPs). Target DNA was co-hybridized with reporter oligos on the GQDs and quencher oligos on AuNPs. This triggers quenching of fluorescence (with excitation/emission peaks at 400 nm/530 nm). On introducing target into the system, fluorescence is quenched by up to 95% by 100 nM concentrations of target oligos having 20 bp. The response to the fliC gene of E. coli O157:H7 increases with the logarithm of the concentration in the range from 0.1 nM to 150 nM. The limit of detection is 1.1 ± 0.6 nM for n = 3. The selectivity and specificity of the assay was confirmed by evaluating the various oligos sequences and PCR product (fliC gene) amplified from genomic DNA of the food samples spiked with E. coli O157:H7. Graphical abstractSchematic representation of fluorometric assay for highly sensitive quantification of Escherichia coli O157:H7 based on fluorescence quenching gene assay for fliC gene of E. coli O157:H7.
A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0-100ng/ml (R(2)=0.992) and log10 (1-10(4) CFU/ml) (R(2)=0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4-37°C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens.
Vibrio cholerae is a human pathogen that causes mild to severe diarrheal illnesses and has major public health significance. Herein, we present a thermostabilized electrochemical genosensing assay combining the use of magnetic beads as a biorecognition platform and gold nanoparticles as a hybridization tag for the detection and quantification of V. cholerae lolB gene single-stranded asymmetric PCR amplicons as an alternative to the time-consuming classical isolation method. This thermostabilized, pre-mixed, pre-aliquoted and ready-to-use magnetogenosensing assay simplified the procedures and permitted the reaction to be conducted at room temperature. The asymmetric PCR amplicons were hybridized to a magnetic bead-functionalized capture probe and a fluorescein-labeled detection probe followed by tagging with gold nanoparticles. Electrochemical detection of the chemically dissolved gold nanoparticles was performed using the differential pulse anodic stripping voltammetry method. The real-time stability evaluation of thermostabilized assay was found to be stable for at least 180 days at room temperature (25-30°C). The analytical specificity of the assay was 100%, while its analytical sensitivity was linearly related to different concentrations of 200-mer synthetic target, purified genomic DNA, and bacterial culture with a limit of detection (LoD) of 3.9nM, 5pg/µl, and 10(3)CFU/ml, respectively. The clinical applicability of the assay was successfully validated using spiked stool samples with an average current signal-to-cut-off ratio of 10.8. Overall, the precision of the assay via relative standard deviation was <10%, demonstrating its reliability and accuracy.
Two Gram-stain-negative, non-fermentative bacterial strains, designated 11-0202(T) and 11-0607, were isolated from soil in South Korea, and four others, LUH 13522, LUH 8638, LUH 10268 and LUH 10288, were isolated from a beet field in Germany, soil in the Netherlands, and sediment of integrated fish farms in Malaysia and Thailand, respectively. Based on 16S rRNA, rpoB and gyrB gene sequences, they are considered to represent a novel species of the genus Acinetobacter. Their 16S rRNA gene sequences showed greatest pairwise similarity to Acinetobacter beijerinckii NIPH 838(T) (97.9-98.4 %). They shared highest rpoB and gyrB gene sequence similarity with Acinetobacter johnsonii DSM 6963(T) and Acinetobacter bouvetii 4B02(T) (85.4-87.6 and 78.1-82.7 %, respectively). Strain 11-0202(T) displayed low DNA-DNA reassociation values (<40 %) with the most closely related species of the genus Acinetobacter. The six strains utilized azelate, 2,3-butanediol, ethanol and dl-lactate as sole carbon sources. Cellular fatty acid analyses showed similarities to profiles of related species of the genus Acinetobacter: summed feature 3 (C16 : 1ω7c, C16 : 1ω6c; 24.3-27.2 %), C18 : 1ω9c (19.9-22.1 %), C16 : 0 (15.2-22.0 %) and C12 : 0 (9.2-14.2 %). On the basis of the current findings, it is concluded that the six strains represent a novel species, for which the name Acinetobacter kookii sp. nov. is proposed. The type strain is 11-0202(T) ( = KCTC 32033(T) = JCM 18512(T)).
A taxonomic study was carried out on strain A-11-3(T), which was isolated from an oil-enriched consortia from the surface seawater of Hong-Deng dock in the Straits of Malacca and Singapore. Cells were aerobic, Gram-negative, non-spore-forming irregular rods. The strain was catalase- and oxidase-negative. It grew on a restricted spectrum of organic compounds, including some organic acids and alkanes. 16S rRNA gene sequence comparisons showed that strain A-11-3(T) was most closely related to the type strains of Alcanivorax jadensis (96.8 % sequence similarity), Alcanivorax borkumensis (96.8 %), Alcanivorax dieselolei (94.8 %), Alcanivorax venustensis (94.2 %) and Alcanivorax balearicus (94.0 %). The predominant fatty acids were C(16 : 0) (31.2 %), C(18 : 1)omega7c (24.8 %), C(18 : 0) (9.6 %), C(12 : 0) (8.3 %), C(16 : 1)omega7c (8.3 %) and C(16 : 0) 3-OH (5.1 %). The G+C content of the genomic DNA was 54.7 mol%. Moreover, the strain produced lipopeptides as its surface-active compounds. According to physiological and biochemical tests, DNA-DNA hybridization results and sequence comparisons of the 16S-23S internal transcribed spacer, the gyrB gene and the alkane hydroxylase gene alkB1, strain A-11-3(T) was affiliated with the genus Alcanivorax but could be readily distinguished from recognized Alcanivorax species. Therefore strain A-11-3(T) represents a novel species of the genus Alcanivorax for which the name Alcanivorax hongdengensis sp. nov. is proposed. The type strain is A-11-3(T) (=CGMCC 1.7084(T)=LMG 24624(T)=MCCC 1A01496(T)).
The taxonomic position of a novel Amycolatopsis strain isolated from a high altitude Atacama Desert subsurface soil was established using a polyphasic approach. The strain, isolate H5T, was shown to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the diamino acid in the cell wall peptidoglycan, arabinose and galactose as diagnostic sugars and MK-9(H4) as the predominant isoprenologue. It also has cultural and morphological properties consistent with its classification in the genus, notably the formation of branching substrate hyphae which fragment into rod-like elements. 16S rRNA gene sequence analyses showed that the strain is closely related to the type strain of Amycolatopsis mediterranei but could be distinguished from this and other related Amycolatopsis strains using a broad range of phenotypic properties. It was separated readily from the type strain of Amycolatopsis balhymycina, its near phylogenetic neighbour, based on multi-locus sequence data, by low average nucleotide identity (92.9%) and in silico DNA/DNA relatedness values (51.3%) calculated from draft genome assemblies. Consequently, the strain is considered to represent a novel species of Amycolatopsis for which the name Amycolatopsis vastitatis sp. nov. is proposed. The type strain is H5T (= NCIMB 14970T = NRRL B-65279T).
A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer-Emmett-Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10-12-1.0×10-2 μM, with a low detection limit of 8.17×10-14 μM (R2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstract Step-by-step DNA biosensor fabrication based on aminated hollow silica spheres.
The quality of a nucleotide-based library such as a synthetic antibody library is highly dependent on the diversity available. Diversity can be generated using degenerate oligonucleotides introduced during gene assembly. Conventional approaches to gene assembly are not efficient for oligonucleotides with long stretches of degeneracy. We propose an efficient alternative for simultaneous introduction of three randomized regions in a synthetic antibody gene via temperature cascading. The strategy takes advantage of DNA reannealing kinetics. The strategy can be adopted for generating diversity of gene inserts during the construction of nucleotide-based libraries.
Beta (β)- thalassaemia is a public health problem in Malaysia. The carrier rate is estimated to be 4.5% by micro-mapping studies particularly among Malays who comprise 53.5% of the population in Malaysia. The common diagnostic method in Malaysia for mutation detection is by amplification refractory mutation system (ARMS). It allows single mutation detection in each reaction but is labour intensive and time consuming when many mutations need to be identified. The purpose of this study was to develop a diagnostic tool for effective mutation detection of beta thalassaemia in Malay patients and compare its efficacy with ARMS-PCR, the current method in use. Methods: Reverse dot blot hybridization (RDBH) technique was incorporated in the development of two strip assays [RDBH-Strip M(6) and RDBH-Strip C(6)] to identify common beta thalassaemia mutations in the Malays. The panels of selected mutations were based on the mutation frequencies in Malaysia reported in previous studies. RDBH-Strip M(6) was applied as step 1 and RDBH-Strip C(6) was applied as step 2 for unidentified mutations. The strips were validated with the gold standard method, ARMS- PCR. Results: One hundred and thirty seven Malay patients with 274 alleles were studied. In Step 1 mutation detection, 238 alleles (86.9%) were identified in 119 of patients by RDBH-Strip M(6). Step 2 resulted in a further detection of 20 alleles in another 10 patients by RDBH-Strip C(6). The combination of both strips resulted in the identification of 258 alleles in 129 (94.6%) of 137 Malay patients. The strip assays were 100% sensitive and specific when compared with ARMS-PCR method. Conclusion: Two strip assays utilising the RDBH technique were developed to identify common β-thalassaemia mutations in Malays. The RDBH Strip M(6) identified 86.9% of the mutations and the RDBH-Strip C (6) detected further 7.3% alleles. This two step strategy was found to be rapid and cost effective for the direct diagnosis of β-thalassaemia mutations in the Malays. The remaining unidentified mutations would require DNA sequencing. It can serve as a specific molecular diagnostic tool for effective diagnosis of
β-thalassaemia mutations in this ethnic group.
Deoxyribonucleic acid (DNA) of twenty chronic myeloid leukemia (CML) and thirty acute lymphoblastic leukemia (ALL) patients were analysed by Southern hybridization. The DNA was digested with BglII and hybridized with a 4.5-kilobase (kb) ph1/bcr-3 DNA probe. All the 20 CML patients showed gene rearrangement within a 5.8-kb segment (the major breakpoint cluster region, M-bcr) of the breakpoint cluster region (bcr) gene of chromosome 22, indicating the presence of the Philadelphia chromosome. M-bcr rearrangement at the bcr gene of chromosome twenty-two was not detected in all the thirty ALL patients (nine adults and twenty-one children) and two normal controls.
Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.
Centromeres are prerequisite for accurate segregation and are landmarks of primary constrictions of metaphase chromosomes in eukaryotes. In melon, high-copy-number satellite DNAs (SatDNAs) were found at various chromosomal locations such as centromeric, pericentromeric, and subtelomeric regions. In the present study, utilizing the published draft genome sequence of melon, two new SatDNAs (CmSat162 and CmSat189) of melon were identified and their chromosomal distributions were confirmed using fluorescence in situ hybridization. DNA probes prepared from these SatDNAs were successfully hybridized to melon somatic and meiotic chromosomes. CmSat162 was located on 12 pairs of melon chromosomes and co-localized with the centromeric repeat, Cmcent, at the centromeric regions. In contrast, CmSat189 was found to be located not only on centromeric regions but also on specific regions of the chromosomes, allowing the characterization of individual chromosomes of melon. It was also shown that these SatDNAs were transcribed in melon. These results suggest that CmSat162 and CmSat189 might have some functions at the centromeric regions.
Six facultatively anaerobic, non-motile lactic acid bacteria were isolated from spontaneous cocoa bean fermentations carried out in Brazil, Ecuador and Malaysia. Phylogenetic analysis revealed that one of these strains, designated M75(T), isolated from a Brazilian cocoa bean fermentation, had the highest 16S rRNA gene sequence similarity towards Weissella fabaria LMG 24289(T) (97.7%), W. ghanensis LMG 24286(T) (93.3%) and W. beninensis LMG 25373(T) (93.4%). The remaining lactic acid bacteria isolates, represented by strain M622, showed the highest 16S rRNA gene sequence similarity towards the type strain of Fructobacillus tropaeoli (99.9%), a recently described species isolated from a flower in South Africa. pheS gene sequence analysis indicated that the former strain represented a novel species, whereas pheS, rpoA and atpA gene sequence analysis indicated that the remaining five strains belonged to F. tropaeoli; these results were confirmed by DNA-DNA hybridization experiments towards their respective nearest phylogenetic neighbours. Additionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved successful for the identification of species of the genera Weissella and Fructobacillus and for the recognition of the novel species. We propose to classify strain M75(T) ( = LMG 26217(T) = CCUG 61472(T)) as the type strain of the novel species Weissella fabalis sp. nov.
A Gram-stain-negative, aerobic, rod-shaped, leaf-associated bacterium, designated JS23T, was isolated from surface-sterilized leaf tissue of an oil palm grown in Singapore and was investigated by polyphasic taxonomy. Phylogenetic analyses based on 16S rRNA gene sequences and 180 conserved genes in the genome of several members of Burkholderiaceae revealed that strain JS23T formed a distinct evolutionary lineage independent of other taxa within the family Burkholderiaceae. The predominant ubiquinone was Q-8. The primary polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminophospholipid. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c /C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c). The size of the genome is 5.36 Mbp with a DNA G+C content of 66.2 mol%. Genomic relatedness measurements such as average nucleotide identity, genome-to-genome distance and digital DNA-DNA hybridization clearly distinguished strain JS23T from the closely related genera Burkholderia, Caballeronia, Mycetohabitans, Mycoavidus, Pandoraea, Paraburkholderia, Robbsia and Trinickia. Furthermore, average amino acid identity values and the percentages of conserved proteins, 56.0-68.4 and 28.2-45.5, respectively, were well below threshold values for genus delineation and supported the assignment of JS23T to a novel genus. On the basis of the phylogenetic, biochemical, chemotaxonomic and phylogenomic evidence, strain JS23T is proposed to represent a novel species of a new genus within the family Burkholderiaceae, for which the name Chitinasiproducens palmae gen. nov., sp. nov., is proposed with the type strain of JS23T (= DSM 27307T=KACC 17592T).
A Gram-negative, filamentous aerobic bacterium designated as strain Mgbs1T was isolated on 12 April 2017 from the subsurface soil and leaf litter substrate at the base of a Koompassia malaccensis tree in a tropical peat swamp forest in the northern regions of the state of Selangor, Malaysia (3° 39' 04.7' N 101° 17' 43.7'' E). Phylogenetic analyses based on the full 16S rRNA sequence revealed that strain Mgbs1T belongs to the genus Chitinophaga with the greatest sequence similarity to Chitinophaga terrae KP01T (97.65 %), Chitinophaga jiangningensis DSM27406T (97.58 %), and Chitinophaga dinghuensis DHOC24T (97.17 %). The major fatty acids of strain Mgbs1T (>10 %) are iso-C15 : 0, C16 : 1 ω5c and iso-C17 : 0 3-OH while the predominant respiratory quinone is menaquinone-7. Strain Mgbs1T has a complete genome size of 8.03 Mb, with a G+C content of 48.5 mol%. The DNA-DNA hybridization (DDH) score between strain Mgbs1T and C. jiangningensis DSM27406T was 15.9 %, while in silico DDH values of strain Mgbs1T against C. dinghuensis DHOC24T and C. terrae KP01T were 20.0 and 19.10% respectively. Concurrently, Average Nucleotide Identity (ANI) scores between strain Mgbs1T against all three reference strains are 73.2 %. Based on the phenotypic, chemotaxonomic, and phylogenetic consensus, strain Mgbs1T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga extrema sp. nov. is proposed (=DSM 108835T=JCM 33276T).
A bacterial strain, designated UTM-3(T), isolated from the rhizosphere soil of Artocarpus integer (cempedak) in Malaysia was studied to determine its taxonomic position. Cells were Gram-stain-negative, non-spore-forming rods, devoid of flagella and gliding motility, that formed yellow-pigmented colonies on nutrient agar and contained MK-6 as the predominant menaquinone. Comparative analysis of the 16S rRNA gene sequence of strain UTM-3(T) with those of the most closely related species showed that the strain constituted a distinct phyletic line within the genus Chryseobacterium with the highest sequence similarities to Chryseobacterium lactis NCTC 11390(T), Chryseobacterium viscerum 687B-08(T), Chryseobacterium tructae 1084-08(T), Chryseobacterium arthrosphaerae CC-VM-7(T), Chryseobacterium oncorhynchi 701B-08(T), Chryseobacterium vietnamense GIMN1.005(T), Chryseobacterium bernardetii NCTC 13530(T), Chryseobacterium nakagawai NCTC 13529(T), Chryseobacterium gallinarum LMG 27808(T), Chryseobacterium culicis R4-1A(T), Chryseobacterium flavum CW-E2(T), Chryseobacterium aquifrigidense CW9(T), Chryseobacterium ureilyticum CCUG 52546(T), Chryseobacterium indologenes NBRC 14944(T), Chryseobacterium gleum CCUG 14555(T), Chryseobacterium jejuense JS17-8(T), Chryseobacterium oranimense H8(T) and Chryseobacterium joostei LMG 18212(T). The major whole-cell fatty acids were iso-C15 : 0 and iso-C17 : 1ω9c, followed by summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1ω7t) and iso-C17 : 0 3-OH, and the polar lipid profile consisted of phosphatidylethanolamine and several unknown lipids. The DNA G+C content strain UTM-3(T) was 34.8 mol%. On the basis of the phenotypic and phylogenetic evidence, it is concluded that the isolate represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium artocarpi sp. nov. is proposed. The type strain is UTM-3(T) ( = CECT 8497(T) = KCTC 32509(T)).
Human papillomavirus (HPV) is well known as an etiological factor for the development of anogenital carcinomas. The aim of our study was to compare the performance of USFDA approved Hybrid II (HCII) Assay and recently introduced DR. HPV Chip Kit for the detection of HPV DNA in clinical cervical scrapings from 40 patients. HPV DNA testing was performed using the automated HCII Assay system and DR. HPV Chip Kit. Taking cytological results as gold standard, it was found that HCII was more sensitive (36.4%) than DR. HPV Chip Kit (18.2%) although specificity was 100% with the latter method. In addition, both these molecular methods had comparable negative and positive predictive values. It was concluded that both HCII and DR. HPV Chip Kit have comparable specificity. However, sensitivity for detection of HPV in clinical samples with HCII is almost double as compared to DR. HPV Chip Kit.
We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis. In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei. and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction. Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns. This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g. B. pseudomallei) but can be used for any bacterial species. The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques. Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.