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

Abstract

Neisseriagonorrhoeae 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.

Nanoparticle-Based Lateral Flow Biosensor Integrated With Loop-Mediated Isothermal Amplification for Rapid and Visual Identification of Chlamydia trachomatis for Point-of-Care Use

“…Loop-mediated isothermal amplification (LAMP), an innovative nucleic acid isothermal amplification technique, was considered as an attractive alternative to traditional NAATs, which has potential applications as a POC testing for its simplicity, rapidity, and easy-to-operate (Notomi et al, 2000 ; Sahoo et al, 2016 ; Shirato, 2019 ). More importantly, it has been widely applied to diagnosis of various pathogens, such as SARS-CoV-2, Neisseria gonorrhoeae , and HIV (Rudolph et al, 2015 ; Chen et al, 2021a , b ). LAMP amplicons can be analyzed with electrophoresis, turbidity, fluorescent dye, and colorimetric indicators (Li et al, 2017 ; Shirato, 2019 ).…”

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

“…Further, the value of the detection limit (45 aM) is equivalent to 10-22 copies µL −1 corresponding to the longest and the shortest supersandwich DNA duplex length of 150 bp and 69 bp, respectively (Figure S3). This detection limit range is superior to those reported by recent studies on visual detection of NG via a multiple cross displacement assay (20 copies/test) and loop-mediated isothermal amplification (LAMP) assay (50 copies/test) [3,6]. In these studies, gold nanoparticles were used as the colorimetric probe.…”

“…Factors that contribute to the high prevalence of gonorrhea include lack of access to testing and treatment, inadequate prevention efforts, and stigma surrounding STIs. Untreated gonorrhea can lead to serious complications, including pelvic inflammatory disease, infertility, neonatal blindness, ectopic pregnancy, and an increased risk of HIV transmission [ 1 , 3 , 4 ]. Another crucial factor to consider is the alarmingly increasing cases of antimicrobial-resistant (AMR) gonorrhea worldwide [ 2 ].…”

“…Biosensors can prove to be essential for improving access to timely and accurate diagnosis, particularly in resource-limited settings where traditional laboratory-based testing may not be readily available. They can allow for the rapid detection of infections including gonorrhea at the point of care (POC), often providing results within minutes, which may help to expedite the treatment and reduce the risk of transmission [ 3 , 6 , 7 ]. In addition, biosensors can reduce the need for expensive and time-consuming laboratory testing, making them an attractive option for both healthcare providers and patients.…”

Visual and rapid identification of Chlamydia trachomatis and Neisseria gonorrhoeae using multiplex loop-mediated isothermal amplification and a gold nanoparticle-based lateral flow biosensor

“…The reaction's efficiency and specificity can be improved by incorporating two extra loop primers (LF and LB) into the LAMP reaction (McGinnis et al, 2021). We have previously used LAMP to detect C. trachomatis and N. gonorrhoeae, respectively, with an LoD as low as 50 copies per test (Chen et al, 2021;Chen et al, 2022). However, it was inadequate for simultaneously detecting these two important pathogens in a single test.…”