Dataset Information

Visual and Rapid Diagnosis of Neisseria gonorrhoeae Using Loop-Mediated Isothermal Amplification Combined With a Polymer Nanoparticle-Based Biosensor in Clinical Application.

ABSTRACT: Neisseria gonorrhoeae is a host-adapted human pathogen that causes sexually transmitted gonorrhea and remains to be a serious global public health challenge, especially in low- and middle-income regions. It is vital to devise a reliable, simple, cost-saving, and easy-to-use assay for detecting the N. gonorrhoeae agent. In the current study, we firstly report a novel approach, loop-mediated isothermal amplification linked with a polymer nanoparticle-based biosensor (LAMP-PNB), that was used for identifying N. gonorrhoeae in clinical samples. The results showed that the LAMP primers based on the orf1 gene were valid for development of the N. gonorrhoeae-LAMP-PNB assay. The detection system with optimal conditions could be performed at a fixed temperature of 64°C for 40 min. The whole process, including genomic DNA preparation (approximately 10 min), LAMP reaction (40 min), and PNB reporting (approximately 2 min), could be accomplished within 60 min. The limit of detection (LoD) of the N. gonorrhoeae-LAMP-PNB assay was 50 copies per test. The specificity of the current assay was 100%, and no cross-reactions to non-N. gonorrhoeae isolates were observed. These results confirmed that the N. gonorrhoeae-LAMP-PNB technique is a reliable, specific, sensitive, rapid, low-cost, and easy-to-use method for detecting gonococci isolates. More importantly, this assay has great potential to develop a point-of-care (POC) testing method in clinical practice, especially in resource-constrained regions.

SUBMITTER: Chen X

PROVIDER: S-EPMC8333867 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission.MethodsA novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples.ResultsThe SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed.ConclusionThe mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.

Project description:Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot meet the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of B. abortus, but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive, and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of B. abortus, which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific B. abortus-LAMP primers targeting the BruAb2_0168 gene was designed by the online LAMP primer design tool. The B. abortus-LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for B. abortus-LAMP-LFIA were determined to be 65°C and 50 min, respectively. The B. abortus-LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of B. abortus. Meanwhile, the detection specificity was 100%, and there was no cross-reactivity for other Brucella members and non-Brucella strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min), and LFIA detection (2-5 min), can be completed in approximately 85 min. Thus, the B. abortus-LAMP-LFIA assay developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.

Dataset Information

Visual and Rapid Diagnosis of Neisseria gonorrhoeae Using Loop-Mediated Isothermal Amplification Combined With a Polymer Nanoparticle-Based Biosensor in Clinical Application.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets