RESUMO
Klebsiella pneumoniae (K. pneumoniae), as an important hospital-acquired bacterium, is responsible for severe morbidity and mortality among the elderly, newborn and immune-compromised people. We established a rcsA gene-based label-free multiple cross displacement amplification (MCDA) assay for rapid, simple and sensitive detection of K. pneumoniae by using lateral flow biosensor (LFB). MCDA reaction was conducted at a fixed temperature (65⯰C) for only 30â¯min, and amplification results were directly indicated using LFB. The results showed that reaction products were detectable from as little as 100â¯fg and 4.8â¯CFU of pure K. pneumoniae templates, and from approximately 480â¯CFU in 1â¯mL of spiked clinical samples. All K. pneumoniae strains examined were positive for label-free MCDA-LFB analysis, and all non-K. pneumoniae strains used in the report were negative for label-free MCDA-LFB assay, indicating the high selectivity of the label free MCDA-LFB assay. Furthermore, to remove false-positive results, the label-free MCDA-LFB assay was supplemented with antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) to eliminate the carryover contamination. Thus, label-free MCDA-LFB assay complemented with AUDG enzyme was a rapid, simple, sensitive and reliable technique for detection of target pathogen, which has the ability to effectively avoid carryover contamination, and can be a valuable tool for "on-site" detection, clinical diagnosis, and primary quarantine purposes.
Assuntos
Técnicas Biossensoriais/métodos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Regiões Antárticas , Técnicas Bacteriológicas/métodos , Sequência de Bases , DNA Bacteriano/análise , Genes Bacterianos/genética , Infecções por Klebsiella/diagnóstico , Sensibilidade e Especificidade , Temperatura , Fatores de Tempo , Uracila-DNA GlicosidaseRESUMO
The authors report on a loop-mediated isothermal amplification (LAMP) scheme that uses antarctic thermally sensitive uracil-DNA-glycosylase (AUDG) for simultaneous detection of nucleic acids and elimination of carryover contamination. It was applied in a lateral flow assay (LFA) format. The assay has attractive features in that it does not require the use of labeled primers or probes, and can eliminate false-positive results generated by unwanted hybridization between two labeled primers or between a labeled primer and probe. LAMP amplification and AUDG digestion are conducted in a single pot, and the application of a closed-tube reaction prevents false-positives due to carryover contamination. The method was applied to the detection of the human pathogen Streptococcus pneumoniaein in pure cultures and spiked blood samples. This LFA can detect S. pneumoniae in pure cultures with a 25 fg.µL-1 detection limit and in spiked blood samples with a 470 cfu.mL-1 detection limit. Conceivably, this assay can be applied to the detection of various other targets if the specific LAMP primers are available. Graphical abstract á .
Assuntos
Técnicas Biossensoriais/métodos , DNA Bacteriano/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Temperatura , Uracila-DNA Glicosidase/metabolismo , DNA Bacteriano/genética , HumanosRESUMO
Vibrio cholerae is an important human pathogen that is responsible for cholera, a severe acute watery diarrhea. In the current study, a multiple cross displacement amplification (MCDA) coupled with amplicon detection by chromatographic lateral flow biosensor (LFB) method (MCDA-LFB) was successfully established and evaluated for the identification of V. cholerae. A set of 10 primers was designed specifically to recognize 10 different regions of the V. cholerae-specific gene ompW. The optimized time and temperature conditions for the MCDA were 30 min and 63°C, respectively. The MCDA-LFB assay correctly identified 31 strains of V. cholerae but did not detect 13 non-cholerae Vibrio strains and 30 non-Vibrio strains. The sensitivity of MCDA-LFB for target pathogen detection in pure culture was 10 fg per reaction. In the case of spiked shrimp samples without enrichment, the limit of detection was 4.1 CFUs per reaction or equivalent to 4.1 × 102 CFU g-1. The whole process, including shrimp homogenates processing (30 min), MCDA reaction (30 min) and results reporting (2 min), could be finished within 65 min. These results show that this assay is suitable for the rapid, sensitive and specific detection of V. cholerae in food, environmental and clinical samples.
Assuntos
Técnicas Biossensoriais/instrumentação , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Vibrio cholerae/genética , Animais , Proteínas da Membrana Bacteriana Externa/genética , Desenho de Equipamento , Microbiologia de Alimentos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Penaeidae/microbiologiaRESUMO
Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3'-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Uracila-DNA Glicosidase/química , Técnicas Biossensoriais , DNA , Primers do DNA , Humanos , Ácidos Nucleicos , Sensibilidade e Especificidade , Tuberculose/diagnóstico , UracilaRESUMO
A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.