Dataset Information

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

ABSTRACT: A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients' serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

SUBMITTER: Liu W

PROVIDER: S-EPMC5000648 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

Liu Wei W Liu Hui-Xin HX Zhang Lin L Hou Xue-Xia XX Wan Kang-Lin KL Hao Qin Q

International journal of molecular sciences 20160803 8

A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully a ...[more]

PMID: 27527151

Similar Datasets

Project description:BACKGROUND: Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. METHODS: A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. RESULTS: The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n=77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. CONCLUSIONS: Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.

Project description:Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

Dataset Information

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

Publications

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets