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1.
Anal Biochem ; 473: 28-33, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25575759

RESUMO

Because loop-mediated isothermal amplification (LAMP) can amplify substantial amounts of DNA under isothermal conditions, its applications for simple genetic testing have attracted considerable attention. A positive LAMP reaction is indicated by the turbidity caused by by-products or by the color change after adding a metallochromic indicator to the reaction solution, but these methods have certain limitations. Leuco crystal violet (LCV), a colorless dye obtained after sodium sulfite treatment of crystal violet (CV), was used as a new colorimetric method for detecting LAMP. LCV is reconverted into CV through contact with double-stranded DNA (dsDNA). Therefore, the positive reaction of LAMP is indicated by color change from colorless to violet. The assay is sensitive enough to detect LAMP products, with a detection limit of 7.1 ng/µl for dsDNA. It is also highly selective to dsDNA, and interference with single-stranded DNA and deoxynucleotide triphosphates (dNTPs) is not observed. LCV facilitates direct colorimetric detection of the main product rather than a by-product of the LAMP reaction; therefore, this method can be used under various reaction conditions such as those with added pyrophosphatase in solution. This colorimetric LAMP detection method using LCV is useful for point-of-care genetic testing given its simplicity.


Assuntos
Colorimetria/métodos , Corantes/química , DNA/análise , DNA/química , Compostos de Tritil/química , DNA/genética , DNA/metabolismo , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Pirofosfatases/metabolismo
2.
J Med Microbiol ; 70(12)2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34878370

RESUMO

Introduction. Non-tuberculosis mycobacterium infections are increasing worldwide, including those caused by rapidly growing mycobacteria (RGM).Gap Statement. The identification of the aetiological agent in the context of infections is essential for the adoption of an adequate therapeutic approach. However, the methods for the rapid distinction of different RGM species are less than optimal.Aim. To develop a nucleic acid chromatography kit to identify clinically common RGM.Methodology. We tried to develop a nucleic acid chromatography kit designed to detect four RGM species (including three subspecies) i.e. Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bolletii (detected as M. abscessus/bolletii) Mycobacterium abscessus subsp. massiliense, Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium peregrinum. The amplified target genes for each species/subspecies using multiplex PCR were analysed using a nucleic acid chromatography assay.Results. Among the 159 mycobacterial type strains and 70 RGM clinical isolates tested, the developed assay correctly identified all relevant RGM without any cross-reactivity or false-negatives. The limits of detection for each species were approximately 0.2 pg µl-1.Conclusion. The rapid and simple nucleic acid chromatography method developed here, which does not involve heat denaturation, may contribute to the rapid identification and treatment of RGM infections.


Assuntos
Cromatografia/métodos , Infecções por Mycobacterium não Tuberculosas , Infecções por Mycobacterium , Micobactérias não Tuberculosas/classificação , Humanos , Infecções por Mycobacterium/diagnóstico , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium abscessus , Ácidos Nucleicos
3.
EBioMedicine ; 64: 103187, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33446475

RESUMO

BACKGROUND: The clinical impact of infection with Mycobacterium (M.) abscessus complex (MABC), a group of emerging non-tuberculosis mycobacteria (NTM), is increasing. M. abscessus subsp. abscessus/bolletii frequently shows natural resistance to macrolide antibiotics, whereas M. abscessus subsp. massiliense is generally susceptible. Therefore, rapid and accurate discrimination of macrolide-susceptible MABC subgroups is required for effective clinical decisions about macrolide treatments for MABC infection. We aimed to develop a simple and rapid diagnostic that can identify MABC isolates showing macrolide susceptibility. METHODS: Whole genome sequencing (WGS) was performed for 148 clinical or environmental MABC isolates from Japan to identify genetic markers that can discriminate three MABC subspecies and the macrolide-susceptible erm(41) T28C sequevar. Using the identified genetic markers, we established PCR based- or DNA chromatography-based assays. Validation testing was performed using MABC isolates from Taiwan. FINDING: We identified unique sequence regions that could be used to differentiate the three subspecies. Our WGS-based phylogenetic analysis indicated that M. abscessus carrying the macrolide-susceptible erm(41) T28C sequevar were tightly clustered, and identified 11 genes that were significantly associated with the lineage for use as genetic markers. To detect these genetic markers and the erm(41) locus, we developed a DNA chromatography method that identified three subspecies, the erm(41) T28C sequevar and intact erm(41) for MABC in a single assay within one hour. The agreement rate between the DNA chromatography-based and WGS-based identification was 99·7%. INTERPRETATION: We developed a novel, rapid and simple DNA chromatography method for identification of MABC macrolide susceptibility with high accuracy. FUNDING: AMED, JSPS KAKENHI.


Assuntos
Antibacterianos/farmacologia , Cromatografia/métodos , DNA/análise , Macrolídeos/farmacologia , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium abscessus/classificação , Filogenia , Reação em Cadeia da Polimerase
4.
Biochim Biophys Acta ; 1768(7): 1848-54, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17498646

RESUMO

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at +2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg(2+) but very high at 10 mM Mg(2+) in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg(2+), and the release of lipoproteins with Asp at +2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at +2 at 2 mM Mg(2+). The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at +2 even at 10 mM Mg(2+), while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Fosfolipídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/metabolismo , Cardiolipinas/metabolismo , Cardiolipinas/farmacologia , Proteínas de Transporte/metabolismo , Lipoproteínas/efeitos dos fármacos , Lipossomos/metabolismo , Magnésio/fisiologia , Chaperonas Moleculares , Proteínas Periplásmicas de Ligação/metabolismo , Fosfatidiletanolaminas/farmacologia , Fosfatidilgliceróis/farmacologia , Proteolipídeos/efeitos dos fármacos , Proteolipídeos/metabolismo
5.
J Biosci Bioeng ; 126(3): 397-403, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29673986

RESUMO

Although nucleic acid amplification test (NAT) is widely used for pathogen detection, rapid NAT systems that do not require special and expensive instruments must be developed in order to enable point of care (POC)-NATs, which contribute to early initiation of treatment. As a POC-NAT system, Kaneka DNA chromatography chip (KDCC), developed using DNA tag-bound primer through modified substance, was shown to be suitable for POC testing, due to the rapid detection time, simple procedures, and low manufacturing costs. However, owing to some modifications in primer, the detection performance and amplification speed were shown to be reduced when using KDCC, counteracting the increased speed of detection. To solve these issues and improve the speed of this NAT system, we investigated a better modification substance for KDCC. Here, azobenzene-modified primers were shown to have the highest amplification speed and detection performance in KDCC, of all modifications tested in this study, showing 10-100-fold lower detection limit but maintaining the same reaction time. Additionally, rapid herpes simplex virus detection system with azobenzene modified primers was developed. We believed that this breakthrough will contribute toward enabling greater utilization of POC-NATs for medical care, especially in developing countries and clinics.


Assuntos
Compostos Azo/química , Cromatografia em Papel/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Cromatografia em Papel/instrumentação , Primers do DNA/química , DNA Bacteriano/química , Humanos , Limite de Detecção , Desnaturação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sistemas Automatizados de Assistência Junto ao Leito
6.
Forensic Sci Int ; 291: 68-75, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30149281

RESUMO

In recent years, the need for analyzing cannabis DNA has increased in order to accommodate the various types of cannabis samples encountered in forensic investigation. This study was aimed to establish a simple and accurate cannabis DNA detection system using DNA chromatography. Two chromatography chip systems with different features were successfully developed. One system (the "four-line version") involves tetraplex PCR amplification, which could be used to detect cannabis DNA and distinguish between drug-type and fiber-type cannabis using the tetrahydrocannabinolic acid synthase gene sequence. The other system was the "three-line version" with triplex amplification, which was specialized to distinguish cannabis from other plants, and had a sensitivity (10fg DNA/reaction) that was 100 times greater than the four-line version. In both versions, no false positives were observed for 60 medicinal plants, and accurate detection could be performed for several simulated forensic samples such as cannabis leaves, buds, stems, roots, seeds, resin, and cannabis leaves blended 1/100 in tobacco. Detection could be performed by the naked eye and only a thermal cycler was required for operation. Thus, DNA chromatography systems for cannabis detection are expected to contribute to the analysis of cannabis DNA in forensic chemistry laboratories without extensive equipment.


Assuntos
Cannabis/genética , Cromatografia/métodos , DNA de Plantas , Primers do DNA , Toxicologia Forense , Limite de Detecção , Reação em Cadeia da Polimerase
7.
FEBS J ; 274(12): 3034-43, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17509078

RESUMO

The LolCDE complex of Escherichia coli belongs to the ATP-binding cassette transporter superfamily and mediates the detachment of lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane. The complex is composed of one copy each of membrane subunits LolC and LolE, and two copies of ATPase subunit LolD. To establish the conditions for reconstituting the LolCDE complex from separately isolated subunits, the ATPase activities of LolD and LolCDE were examined under various conditions. We found that both LolD and LolCDE were inactivated on incubation at 30 degrees C in a detergent solution. ATP and phospholipids protected LolCDE, but not LolD. Furthermore, phospholipids reactivated LolCDE even after its near complete inactivation. LolD was also protected from inactivation when membrane subunits and phospholipids were present together, suggesting the phospholipid-dependent reassembly of LolCDE subunits. Indeed, the functional lipoprotein-releasing machinery was reconstituted into proteoliposomes with E. coli phospholipids and separately purified LolC, LolD and LolE. Preincubation with phospholipids at 30 degrees C was essential for the reconstitution of the functional machinery from subunits. Strikingly, the lipoprotein-releasing activity was also reconstituted from LolE and LolD without LolC, suggesting the intriguing possibility that the minimum lipoprotein-releasing machinery can be formed from LolD and LolE. We report here the complete reconstitution of a functional ATP-binding cassette transporter from separately purified subunits.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Fosfolipídeos/química , Subunidades Proteicas/química
8.
Harmful Algae ; 51: 97-106, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28003065

RESUMO

In this study, the Kaneka DNA chromatography chip (KDCC) for the Alexandrium species was successfully developed for simultaneous detection of five Alexandrium species. This method utilizes a DNA-DNA hybridization technology. In the PCR process, specifically designed tagged-primers are used, i.e. a forward primer consisting of a tag domain, which can conjugate with gold nanocolloids on the chip, and a primer domain, which can anneal/amplify the target sequence. However, the reverse primer consists of a tag domain, which can hybridize to the solid-phased capture probe on the chip, and a primer domain, which can anneal/amplify the target sequence. As a result, a red line that originates from gold nanocolloids appears as a positive signal on the chip, and the amplicon is detected visually by the naked eye. This technique is simple, because it is possible to visually detect the target species soon after (<5min) the application of 2µL of PCR amplicon and 65µL of development buffer to the sample pad of the chip. Further, this technique is relatively inexpensive and does not require expensive laboratory equipment, such as real-time Q-PCR machines or DNA microarray detectors, but a thermal cycler. Regarding the detection limit of KDCC for the five Alexandrium species, it varied among species and it was <0.1-10pg and equivalent to 5-500 copies of rRNA genes, indicating that the technique is sensitive enough for practical use to detect several cells of the target species from 1L of seawater. The detection sensitivity of KDCC was also evaluated with two different techniques, i.e. a multiplex-PCR and a digital DNA hybridization by digital DNA chip analyzer (DDCA), using natural plankton assemblages. There was no significant difference in the detection sensitivity among the three techniques, suggesting KDCC can be readily used to monitor the HAB species.

9.
Front Microbiol ; 6: 1548, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26793181

RESUMO

We have developed a novel Neisseria meningitidis serogroup-specific loop-mediated isothermal amplification (LAMP) assay for six of the most common meningococcal serogroups (A, B, C, W, X, and Y). The assay was evaluated using a set of 31 meningococcal LAMP assay positive cerebrospinal fluid (CSF) specimens from 1574 children with suspected meningitis identified in prospective surveillance between 1998 and 2002 in Vietnam, China, and Korea. Primer specificity was validated using 15 N. meningitidis strains (including serogroups A, B, C, E, W, X, Y, and Z) and 19 non-N. meningitidis species. The N. meningitidis serogroup LAMP detected down to ten copies and 100 colony-forming units per reaction. Twenty-nine CSF had N. meningitidis serogroup identified by LAMP compared with two CSF in which N. meningitidis serogroup was identified by culture and multi-locus sequence typing. This is the first report of a serogroup-specific identification assay for N. meningitidis using the LAMP method. Our results suggest that this assay will be a rapid, sensitive, and uniquely serogroup-specific assay with potential for application in clinical laboratories and public health surveillance systems.

10.
J Med Microbiol ; 64(11): 1335-1340, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26338293

RESUMO

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis complex (MTC), remains one of the leading causes of death in the world. In Korea, the current prevalence of multidrug-resistant TB (MDR-TB) poses a major problem. The most common method for diagnosing TB in developing countries is sputum smear microscopy; however, the sensitivity of this test is relatively low and it usually requires well-trained laboratory staff. Cultures of MTC require up to several weeks in sophisticated facilities, such as Biosafety Level 3. Effective diagnostic techniques are necessary to control TB. In Korea, we evaluated a loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene (TB-hspX-LAMP) of MTC. For clinical evaluation, culture confirmation, smear microscopy and TB-hspX-LAMP were performed on 303 sputum specimens obtained from suspected TB patients in Korea. The sensitivity, specificity, positive predictive value and negative predictive value of TB-hspX-LAMP were 71.1, 98.8, 91.4 and 95.1%, respectively, compared with TB culture, which is the gold standard for diagnosis of TB. In contrast, the comparable values of smear microscopy were 24.4, 98.1, 68.8 and 88.2%, respectively. Therefore, we concluded that TB-hspX-LAMP was superior to the use of smear microscopy for the detection of MTC in sputum specimens in clinical settings in Korea.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , República da Coreia , Tuberculose Pulmonar/diagnóstico
11.
PLoS One ; 10(4): e0122922, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25853422

RESUMO

BACKGROUND: Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients' cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF). METHODOLOGY/PRINCIPAL FINDINGS: We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively. CONCLUSIONS/SIGNIFICANCE: Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.


Assuntos
Líquido Cefalorraquidiano/microbiologia , DNA Bacteriano/isolamento & purificação , Meningite Meningocócica/líquido cefalorraquidiano , Neisseria meningitidis/isolamento & purificação , Adulto , Sequência de Bases , Criança , DNA Bacteriano/genética , Feminino , Humanos , Lactente , Masculino , Meningite Meningocócica/microbiologia , Neisseria meningitidis/patogenicidade
12.
Nucl Med Biol ; 30(3): 327-34, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12745024

RESUMO

In vivo metabolism of [(188)Re]tricarbonyl(carboxycyclopentadienyl)rhenium ([(188)Re]CpTR-COOH) and its glycine conjugate ([(188)Re]CpTR-Gly) was investigated to estimate the applicability of cyclopentadienyltricarbonylrhenium (CpTR) compounds to (186/188)Re-labeling reagents for polypeptides and peptides. Both [(188)Re]CpTR derivatives were stable after incubation in a buffered-solution and in murine plasma at 37 degrees C for 6 h. Plasma protein binding was hardly observed with the two derivatives. However, different biodistribution and metabolic fates were observed with the two CpTR derivatives. While more lipophilic [(188)Re]CpTR-COOH was excreted by both hepatobiliary and urinary excretion, the majority of less lipophilic [(188)Re]CpTR-Gly was excreted by urinary excretion. In addition, while [(188)Re]CpTR-Gly was rapidly excreted into urine as its intact structure, [(188)Re]CpTR-COOH was metabolized to more hydrophilic compounds including its glycine conjugate, [(188)Re]CpTR-Gly. Renal excretion of [(188)Re]CpTR-Gly was significantly reduced in probenecid retreated mice. The present studies reinforced that CpTR core remained stable under biological environment. CpTR-COOH was partially recognized as an aromatic acid and was metabolized as such. However, glycine conjugation rendered CpTR-COOH hydrophilic enough to be excreted into urine without further metabolism. These findings suggested that radiolabeling reagents that liberate [(186/188)Re]CpTR-Gly from covalently conjugated (186/188)Re-labeled polypeptides and peptides by the action of renal brush border enzymes would be useful to reduce renal radioactivity levels.


Assuntos
Compostos Organometálicos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Camundongos , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacocinética , Distribuição Tecidual
13.
Mol Microbiol ; 62(4): 1064-75, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17038124

RESUMO

The LolCDE complex of Escherichia coli belongs to the ABC transporter superfamily and initiates the lipoprotein sorting to the outer membrane by catalysing their release from the inner membrane. LolC and/or LolE, membrane subunits, recognize lipoproteins anchored to the outer surface of the inner membrane, while LolD hydrolyses ATP on its inner surface. We report here that ligand-bound LolCDE can be purified from the inner membrane in the absence of ATP. Liganded LolCDE represents an intermediate of the release reaction and exhibits higher affinity for ATP than the unliganded form. ATP binding to LolD weakens the interaction between LolCDE and lipoproteins and causes their dissociation in a detergent solution, while lipoprotein release from membranes requires ATP hydrolysis. Liganded LolCDE thus reveals molecular events brought about through ATP binding and hydrolysis. LolCDE is the first example of an ABC transporter purified with tightly bound native substrates. A single molecule of lipoprotein is found to bind per molecule of the LolCDE complex.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos/genética , Membrana Celular/metabolismo , Detergentes , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Ligantes , Lipoproteínas/química , Mutação , Proteolipídeos/metabolismo , Soluções
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