Loop-Mediated Isothermal Amplification Assay Using Gold Nanoparticles for Detecting Treponema pallidum subspp. pallidum.

BACKGROUND: Venereal syphilis in humans is caused by Trepenoma pallidum subspp. pallidum. A study has shown that 30,302 individuals in Thailand had syphilis in 2020, with a male-to-female ratio of 1:0.8 and the highest incidence rate at ages between fifteen and twenty-four. METHODS: This research aimed to develop a loop-mediated isothermal amplification assay using gold nanoparticles (LAMP-AuNPs). Analytical sensitivity, diagnostic specificity, accuracy, and predictive values for each technique are provided. RESULTS: The diagnosis sensitivities of polymerase chain reaction using agarose gel electrophoresis (PCR-AGE), loop-mediated isothermal amplification assay using agarose gel electrophoresis (LAMP-AGE), and LAMP-AuNPs were 116 ng/µL, 11.6 ng/µL, and 11.6 ng/µL, respectively. We evaluated the analytical specificity using PCR and a LAMP-based assay, and there was no cross-reactivity to Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy humans. After analyzing 400 serum samples of patients suspected of syphilis, the LAMP-AGE and LAMP-AuNPs assays displayed 100% diagnostic sensitivity scores, 91% diagnostic specificity scores, 95.5% accuracy rates, 100% positive predictive values (PPVs), and 91% negative predictive values (NPVs), the positive likelihood ratio (LR+) was 11.11, while the negative likelihood ratio (LR-) was 0. Conversely, for PCR assays displayed 100% diagnostic sensitivity scores, 94.5% diagnostic specificity scores, 97.25% accuracy rates, 100% PPVs, and 94.5% NPVs, LR+ was 18.18, and LR- was 0. CONCLUSIONS: The LAMP-AuNPs technique demonstrates rapidity, affordability, and convenience, rendering it well-suited for point-of-care applications in the diagnosis, prevention, and management of pathogenic infections.

Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Molecular characterization of extended-spectrum cephalosporin and fluoroquinolone resistance genes in Salmonella and Shigella isolated from clinical specimens in Thailand.

Antimicrobial resistance of Salmonella and Shigella has become a major clinical and public health problem. The incident of co-resistance to third generation cephalosporins and fluoroquinolone is a serious therapeutic issue in Thailand. The present study aimed to investigate the antimicrobial resistance and molecular character of clinical Shigella and Salmonella isolates. A total of 33 Salmonella and 53 Shigella cefotaxime-resistant isolates were collected from human clinical cases in Thailand during the period from 2011-2018. The antimicrobial susceptibility of Salmonella and Shigella was determined by the disk diffusion method, and extended-spectrum beta-lactamase (ESBL) production was characterized by the double-disk synergy test. Genotype characterization was performed by PCR and DNA sequencing. Thirty-two (97.0%) and fifty-two (98.1%) isolates of cefotaxime-resistant Salmonella and Shigella, respectively, were identified as ESBL producers. Shigella sonnei (4 isolates), Salmonella serovar 4,5,12:i:- (6 isolates), Salmonella serovar Agona (2 isolates) and Salmonella serovar Rissen (2 isolates) showed co-resistance to ciprofloxacin and cefotaxime or ceftriaxone. The combination of bla CTX-M-15 plus other ESBL and/or AmpC ß-lactamase genes was the most dominant of the genotype patterns in ESBL-producing isolates. The plasmid harbouring the aac(6')-Ib-cr gene and mutations of gyrA (S83F, D87Y or D87G) and parC (T57S) genes was found in 2 ESBL-producing Salmonella isolates. Three Shigella sonnei isolates harboured mutations in gyrA (S83L, D87Y or D87G), and only one Shigella sonnei phase I isolate showed mutations in both gyrA (S83L and D87G) and parC (S80I) genes. Among these clinical Shigella sonnei isolates, qnrS determinants were identified. Production of ESBLs is an important mechanism for resistance to extended-spectrum cephalosporins in Salmonella and Shigella. The emergence of a decreased susceptibility to extended-spectrum cephalosporins and fluoroquinolone in ESBL-producing isolates has important clinical and therapeutic implications.

In-Situ Monitoring of Real-Time Loop-Mediated Isothermal Amplification with QCM: Detecting Listeria monocytogenes.

Functionalized DNA sequences are promising sensing elements to combine with transducers for bio-sensing specific target microbes. As an application example, this paper demonstrates in situ detection of loop-mediated isothermal amplification products by hybridizing them with thiolated-ssDNA covalently anchored on the electrodes of a quartz crystal microbalance (QCM). Such hybridization leads to a frequency signal, which is suitable for monitoring real-time LAMP amplification based on mass-sensing: it detects interactions between the complementary nucleobases of LAMP products in solution and the thiolated-ssDNA probe sequence on the gold surface. Target DNA LAMP products cause irreversible frequency shifts on the QCM surfaces during hybridization in the kHz range, which result from both changes in mass and charge on the electrode surface. In order to confirm the LAMP assay working in the QCM sensing system at elevated temperature, the sky blue of positive LAMP products solution was achieved by using the Hydroxy Naphthol Blue (HNB) and agarose gel electrophoresis. Since on-QCM sensing of DNA hybridization leads to irreversible sensor responses, this work shows characterization by X-ray photoelectron spectroscopy (XPS) core spectra of S2p, N1s, Mg1s, P2p and C1s. XPS results confirmed that indeed both DNA and by-products of LAMP attached to the surface. Listeria monocytogenes DNA served to study in-situ detection of amplified LAMP products on DNA-functionalized surfaces.

Molecular Characterization of Cephalosporin and Fluoroquinolone Resistant Salmonella Choleraesuis Isolated from Patients with Systemic Salmonellosis in Thailand.

The antimicrobial resistance of nontyphoidal Salmonella has become a major clinical and public health problem. Southeast Asia has a high level of multidrug-resistant Salmonella and isolates resistant to both fluoroquinolone and third-generation cephalosporins. The incidence of co-resistance to both drug classes is a serious therapeutic problem in Thailand. The aim of this study was to determine the antimicrobial resistance patterns, antimicrobial resistance genes and genotypic relatedness of third-generation cephalosporins and/or fluoroquinolone-resistant Salmonella Choleraesuis isolated from patients with systemic salmonellosis in Thailand. Antimicrobial susceptibility testing was performed using the agar disk diffusion method, and ESBL production was detected by the combination disc method. A molecular evaluation of S. Choleraesuis isolates was performed using PCR and DNA sequencing. Then, a genotypic relatedness study of S. Choleraesuis was performed by pulse field gel electrophoresis. All 62 cefotaxime-resistant S. Choleraesuis isolates obtained from 61 clinical specimens were multidrug resistant. Forty-four isolates (44/62, 71.0%) were positive for ESBL phenotypes. Based on the PCR sequencing, 21, 1, 13, 23, 20 and 6 ESBL-producing isolates harboured the ESBL genes blaCTX-M-14, blaCTX-M-15, blaCTX-M-55, blaCMY-2, blaACC-1 and blaTEM-1, respectively. This study also found that nine (9/62, 14.5%) isolates exhibited co-resistance to ciprofloxacin and cefotaxime. All of the co-resistant isolates harboured at least one PMQR gene. The qnr genes and the aac(6')-Ib-cr gene were the most prevalent genes detected. The QRDR mutation, including the gyrA (D87Y and D87G) and parC (T57S) genes, was also detected. PFGE patterns revealed a high degree of clonal diversity among the ESBL-producing isolates.

First Draft Genome Sequence of Thermophilic Laceyella tengchongensis BKK01, Isolated from Municipal Solid Waste in Thailand.

Laceyella tengchongensis BKK01 is a thermophilic bacterium isolated from municipal solid waste. The genome of L. tengchongensis BKK01 includes a gene putatively encoding gramicidin S synthase. Gramicidin S has antibiotic activity against some bacteria and fungi. The newly sequenced 3.44-Mb draft genome of L. tengchongensis BKK01 will shed some light on the biosynthesis of gramicidin S.

Colorimetric aptasensor for detecting Salmonella spp., Listeria monocytogenes, and Escherichia coli in meat samples.

In this study, we successfully developed a simple and rapid method for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli using gold nanoparticles and the aptamer aptasensor. We screened 25 specific DNA aptamer candidates against these pathogens using whole-cell Systematic Evolution of Ligands by EXponential enrichment. Among them, Ap6 was selected due to its low energy minimization values of -12.25 and -27.67 kcal/mol derived from MFold and RNAFold analysis, respectively. The assay presented in this study allowed the visual colorimetric detection of labeled colloidal gold nanoparticles as well as determination of UV absorbance at 625 and 525 nm under optimized conditions. The detection limit of this aptasensor was as less as 105 CFU/ml. A random investigation of 50 meat samples, including ham and chicken sausages, collected from the local market revealed 96% accuracy, 96% specificity, and 100% sensitivity of the assay. The colorimetric aptasensor can accomplish one-step detection without pre-culture, DNA extraction, and amplification. Hence, it is an easy, rapid, specific, and qualitative assay that can be used as a point-of-care testing to directly detect multiplex foodborne pathogens.

Development of a duplex lateral flow dipstick test for the detection and differentiation of Listeria spp. and Listeria monocytogenes in meat products based on loop-mediated isothermal amplification.

Listeria spp. are a group of gram-positive bacteria consisting of 20 species. Among them, Listeria monocytogenes is one of the major species that infects humans since it contaminates raw fruits, vegetables, and many others food products. The conventional methods for the detection of Listeria spp. and L. monocytogenes are time-consuming, taking 5-7 days. Herein, a duplex lateral flow dipstick (DLFD) test combined with loop-mediated isothermal amplification (LAMP) was developed for the identification of Listeria spp. and L. monocytogenes within approximately 45 min with the optimized LAMP reaction times at 63 °C. Under the optimized conditions, the method detection limits (MDL) with reference to genomic DNA and pure culture were 900 femtograms (fg) and 20 cfu/mL, respectively. The LAMP-DLFD showed no cross-reactivity with eighteen - other pathogenic bacteria such as Salmonella spp., Staphylococcus aureus, Escherichia coli, Campylobacter coli, C. jejuni, Enterococcus faecalis, Vibrio cholerae, V. parahaemolyticus, Pseudomonas aeruginosa, Shigella dysenteriae, S. flexneri, Bacillus cereus, Lactobacillus acidophilus, L. casei and Pediococcus pentosaceus. Among 100 samples of food products, LAMP-DLFD demonstrated 100% accuracy when compared to other standard detection methods, such as ISO11290-1, enzyme-linked fluorescent assay (ELFA) technology (VIDAS) and PCR. In conclusion, LAMP-DLFD proved to be highly specific and sensitive assays for screening detection of Listeria spp. and L. monocytogenes.

Draft Genome Sequence of Multidrug-Resistant Proteus mirabilis CKTH01, Isolated from Raw Chicken Meat.

Proteus mirabilis CKTH01 is a pathogenic bacterium isolated from raw chicken meat. The genome sequence of P. mirabilis CKTH01 contains genes encoding multidrug efflux pumps, which are the virulence factors of the antibiotic-resistant bacterium. This 3.98-Mb draft genome sequence of P. mirabilis CKTH01 will contribute to the understanding of the distribution of multidrug-resistant P. mirabilis in raw chicken meat at the open markets.

Rapid Colorimetric Assay for Detection of Listeria monocytogenes in Food Samples Using LAMP Formation of DNA Concatemers and Gold Nanoparticle-DNA Probe Complex.

Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity, and accuracy were 100, 90.20, and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

QCM-based rapid detection of PCR amplification products of Ehrlichia canis.

Ehrlichia canis is an intracellular parasitic bacterium and arthropod-borne pathogen that receives growing attention, because it leads to increasing morbidity and mortality in animals. It does so by causing canine monocytotropic ehrlichiosis (CME). Infected canines may lack obvious clinical signs and stay in chronic stage. Herein we report a rapid screening method based on PCR assay combined with quartz crystal microbalance (QCM) to design a DNA sensor for detecting E. canis in early stages of infection. The test relies on DNA amplification of target nucleotide sequences via PCR followed by detecting DNA-DNA hybridization using QCM. The approach did not result in any cross-hybridization toward other blood bacteria or parasites in dogs, such as Anaplasma platys, Babesia canis and Trypanosoma spp, but turned out selective for the target species. The limit of detection of QCM was as low as 4.1 × 109 molecules/µl of 289 bp E. canis PCR products corresponding to 22 copy numbers/µl of E. canis. Furthermore, the technique is also simple, does not require complicated equipment and can in principle be reused.

Development and evaluation of a loop-mediated isothermal amplification combined with au-nanoprobe assay for rapid detection of Mycobacterium tuberculosis.

Loop-mediated isothermal amplification (LAMP) has been proposed as an inexpensive and easy to perform assay for molecular diagnostics. We present a novel strategy for the detection of LAMP amplicons derived from Mycobacterium tuberculosis by the use of Au-nanoprobes. When applied to a total of 93 clinical specimens, the LAMP assay demonstrated sensitivity and specificity higher than that of polymerase chain reaction and culture. The Au-nanoprobe augmented LAMP test platform with its advantages of robust reagents and a simple colorimetric detection method can be adapted easily for the rapid detection of other infectious disease agents at a low cost.

Autologous Fibrin-Base Scaffold for Chondrocytes and Bone Marrow Mesenchymal Stem Cells Implantation: The Development and Comparison to Conventional Fibrin Glue.

Objective: The authors developed the autologous fibrin-base scaffold for chondrocytes and bone marrow mesenchymal stem cells (BM-MSCs) implantation and evaluated cells viability in autologous fibrin-base scaffold comparing to commercial fibrin glue. Material and Method: The chondrocytes and BM-MSCs were seeded into autologous fibrin-base scaffold and commercial fibrin glue. The cell viability and proliferation were evaluated at 1 and 7 days. The histology were evaluated with hematoxylineosin (H&E) staining and cartilaginous matrices formation with Alcian blue, Saffanin-0, Toluidine blue, and Collagen type II staining at 6 weeks. The fixation of the scaffolds was observed. Results: The chondrocytes and BM-MSCs could not survive in commercial fibrin glue. The chondrocytes and BM-MSCs in autologous fibrin-base scaffold could proliferate and synthesize the cartilaginous matrices on Alcian blue, Saffanin-0, Toluidine blue, and Collagen type II staining at 6 weeks. The fixation strength is excellent. Conclusion: The developed autologous fibrin-base scaffold can be used as the scaffold for chondrocytes and BM-MSCs implantation with potential to implant chondrocytes and BM-MSCs arthroscopically.

Outbreak of Occupational Brucellosis in a Laboratory Technician at Her Royal Highness Princess Sirindhorn Medical Center, Srinakharinwirot University, Thailand.

Objective: To document laboratory transmission of brucellosis and identify the likely mechanism of transmission of brucellosis at Her Royal Highness (HRH) Princess Sirindhorn Medical Center, Thailand. Material and Method: Using small subunit ribosomal RNA (rRNA) sequencing technique to analyze Brucella melitensis cultured from the first 2 patients of the hospital and an infected laboratory technician, and using brucellosis serologic test to rule out infections in all other involved technicians. Results: We had encountered the first 2 cases of brucellosis. Both had infected from community exposure with goat. The first case had pancreatic abscess and spinal bone involvement with a positive blood culture. The second case presented with fever of unknown origin and had a positive blood culture. A few weeks later, 1 of our laboratory technicians presented with fever, myalgia and fatigue. Blood culture grew B. melitensis. He never had any associated community-acquired risk factors for brucellosis. The presumed mechanism of transmission was an inhalation while taking photographs of the bacterial plate of the first patient. B. melitensis identified from our laboratory technician and both patients were analyzed based on 16S-23S rRNA intergenic transcribed spacer (ITS) region. Results of 16S-23S rRNA ITS sequence testing confirmed a match from all patients and laboratory technician's isolate. All other 10 potentially exposed laboratory technicians were asymptomatic. A brucellosis serologic test was negative in all non-infected technicians but was only positive in the 1 infected technician. Conclusion: This is the first report in Thailand of occupational brucellosis transmitted in microbiologic laboratory. The most likely mechanism is air-borne inhalation of bacterial organisms on culture media in the absence of adequate precautions. Laboratory technicians should handle Brucella cultivation with caution utilizing appropriate measures to prevent inhalation.

Comparative Studies on Nucleic Acid Based Biosensors for Identification of Filarial Nematode.

Background: The microfilarial nematodes, found mainly in blood circulation, form a special group of human and veterinary parasitic diseases. Recently, the rapid nucleic acid based biosensors (NABs) have been established in our laboratory for the employment of a point-of-care diagnostic test. Herein, NABs involved in the exploitation of lateral flow dipstick (LFD), gold nanoparticle (AuNP) and turbidity in combination with PCR and LAMP amplification for detection of filarial nematodes. The validation of each NABs was investigated in comparison to that of standard detection methods using the same unknown blood specimens. Objective: To compare the sensitivity, specificity and accuracy of NABs with standard detection methods. Material and Method: In this study, the microfilariae of Dirofilaria immitis was used as the representative model for filarial nematode. The PCR and LAMP primers were designed and synthesized according to the specific nucleotide regions of a small subunit gene of the parasite as well as the DNA probes. The fifty unknown blood samples were sent as a gift from Prasu Arthorn Animal Hospital, Faculty of Veterinary Science, Mahidol University. The samples were tested by using PCR, PCRLFD, PCR-AuNP, LAMP, LAMP-LFD, LAMP-AuNP, LAMP-turbidity and film blood smear. The efficacy of NABs was compared to a standard film blood smear in terms of sensitivity, specificity and accuracy. Results: Upon detection of fifty unknown blood samples, LAMP-LFD assay presented 100% of sensitivity, specificity and accuracy. The data revealed that sensitivity, specificity and accuracy of NABs varied from 66.67-100.00% when compared to a film blood smear stained with Giemsa dye. The data clearly indicated that LAMP-LFD was a preferred choice for the use as a point-of-care NABs. Conclusion: NABs were highly sensitive performing as a selective diagnostic tool that could be applied particularly as the rapid screening tests for filarial nematodes hence as a recommended epidemiological survey.

Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris.

A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%-64% identity to 23 sequences from actinomycetes (23 α/ß-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/ß-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0-8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 µmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2-C6), displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM(-1) · S(-1)). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

A piezoelectric-based immunosensor for high density lipoprotein particle measurement.

A piezoelectric-based immunosensor was developed for high density lipoprotein particle (HDL-P) measurement. Monoclonal anti-human apolipoprotein A1 antibody was used as a specific binding molecule for the major apolipoprotein of HDL-P. This sensing element was fabricated by immobilizing the anti-human apolipoprotein A1 on a 12 MHz AT-cut quartz crystal via a 3-mercaptopropionic acid (MPA) self-assembled monolayer. The frequency shift from the mass change of the antigen-antibody binding refers to the amount of HDL-P. The optimal antibody immobilization was performed to achieve the maximum potential of the antibody. The appropriate quantity and immobilization time of the antibody were 0.1 mg ml(-1) and 90 minutes, respectively. The immobilized antibody in the HDL-P immunosensor accomplished perfect binding with HDL-P within 60 minutes. The dose-response curve for HDL-P showed a linear response from 0.21 to 2.50 mg protein per ml equivalent to 0.40 × 10(10) to 3.65 × 10(10) particles per µl without significant interference from other lipoproteins. The intra- and inter-assay imprecision (CV) were 7.8 and 18.5%, respectively. The analytical accuracy of this measurement was 96.29-96.31%. The HDL-P concentration obtained from the sensor revealed a 2.05 mg protein per ml with 0.26 mg protein per ml of expanded uncertainty at the 95% confidence level. This immunosensor gave an assay result which correlated with the homogeneous enzymatic colorimetric assay (R(2) = 0.902).

Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples.

Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92   % and the specificity was 100   % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.

Expression and characterization of Cu/Zn superoxide dismutase from Wuchereria bancrofti.

The Cu/Zn superoxide dismutase gene from Wuchereria bancrofti (Cu/Zn WbSOD) was isolated by PCR using degeneracy primers. The complete Cu/Zn WbSOD consisted of 1,032 nucleotides containing 4 exons (477 nucleotides) and 3 introns. The molecular phylogenetic analysis of the Cu/Zn WbSOD gene in comparison with those from other organisms revealed that the gene was classified in the same clade to those of filarial Brugia malayi and Brugia pahangi (bootstrap value at 90). The nucleotide and deduced amino acid sequences of Cu/Zn WbSOD exhibited the similarity to those of intracellular Cu/Zn SOD of B. malayi and B. pahangi. The amino acid comparison of Cu/Zn WbSOD to others revealed that the binding sites and active sites were conserved. The expression of this gene yielded 16.366 kDa in size. After Ni-IDA column purification, the enzyme showed specific activity of 8.5 U/mg and 42.1% yield. The enzyme activity was inhibited when 6 mM KCN was added.

Characterization of thermophilic halotolerant Aeribacillus pallidus TD1 from Tao Dam Hot Spring, Thailand.

The bacterial strain TD1 was isolated from Tao Dam hot spring in Thailand. Strain TD1 was Gram positive, rod-shaped, aerobic, motile, and endospore forming. The cell was 2.0-40 µm in length and about 0.4 µm in diameter. The optimum growth occurred at 55-60 °C and at pH 7-8. Strain TD1 was able to grow on medium containing up to 10% NaCl. The DNA G+C content was 38.9 mol%. The cellular fatty acid content was mainly C(16:0), which comprised 25.04% of the total amount of cellular fatty acid. 16S rDNA showed 99% identity to Aeribacillus pallidus DSM 3670(T). Bayesian tree analysis strongly supported the idea that strain TD1 is affiliated with genus Aeribacillus, as Aeribacillus pallidus strain TD1. Although the 16S rDNA of A. pallidus strain TD1 is similar to that of A. pallidus DSM 3670(T), some physiological properties and the cellular fatty acid profiles differ significantly. A. pallidus strain TD1 can produce extracellular pectate lyase, which has not been reported elsewhere for other bacterial strains in the genus Aeribacillus. A. pallidus strain TD1 may be a good candidate as a pectate lyase producer, which may have useful industrial applications.