Establishment and application of a rapid visual diagnostic method for Streptococcus agalactiae based on recombinase polymerase amplification and lateral flow strips.

This study aims to establish a rapid diagnostic method for Streptococcus agalactiae (GBS) based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). The best primer pairs designed by SIP gene were screened according to the basic RPA reaction, then the probe was designed. The reaction condition was optimized based on the color development of the LFS detection line. To ascertain the reaction specificity, 10 common clinical pathogens and 10 clinical specimens of GBS were tested. Furthermore, the reaction sensitivity was assessed by utilizing a tenfold gradient dilution of GBS genomic DNA as templates. RPA-LFS method was compared to the qPCR assay and biochemical culture method for the Kappa consistency test. The RPA-LFS technique was able to complete the amplification process within 30 min and the results were observed on lateral flow strips. The method is highly sensitive, with a minimum detection limit of 1.31 ng for GBS. The RPA-LFS method showed consistent accuracy of results compared to qPCR and the culture-biochemical method. The establishment of this method is conducive to the development of on-site immediate detection, which can provide information for the timely development of a reasonable antimicrobial treatment plan, and has a greater potential for clinical application.

Simultaneous detection of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) using recombinase polymerase amplification.

Bovine viral diarrhea virus (BVDV) is considered to be the most common agent of severe diarrhea in cattle worldwide, causing fever, diarrhea, ulcers, and abortion. Bovine herpesvirus 1 (BoHV-1) is also a major bovine respiratory disease agent that spreads worldwide and causes extensive damage to the livestock industry. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method with the advantages of high efficiency, rapidity and sensitivity, which has been widely used in the diagnosis of infectious diseases. A dual RPA assay was developed for the simultaneous detection of BVDV and BoHV-1. The assay was completed at a constant temperature of 37 °C for 30 min. It was highly sensitive and had no cross-reactivity with other common bovine viruses. The detection rate of BVDV RPA in clinical samples (36.67%) was higher than that of PCR (33.33%), the detection rate of BoHV-1 RPA and PCR were equal. Therefore, the established dual RPA assay for BVDV and BoHV-1 could be a potential candidate for use as an immediate diagnostic.

Development of a real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primers for rapid detection of rubella virus.

Rubella virus infection during early pregnancy sometimes causes severe birth defects termed congenital rubella syndrome. Although there are safe and effective live-attenuated vaccines, rubella has only been certified as eliminated in the Americas within the six World Health Organization regions. Rubella remains an endemic disease in many regions, and outbreaks occur wherever population immunity is insufficient. There are two main methods for diagnosis of rubella: detection of anti-rubella IgM antibodies by enzyme immunoassay and detection of the viral genome by real-time RT-PCR. Both of these methods require substantial time and effort. In the present study, a rapid rubella detection assay using real-time fluorescent reverse transcription loop-mediated isothermal amplification with quenching primers was developed. The time required for the new assay was one-half that required for a real-time RT-PCR assay. The assay had 93.6% positive percent agreement and 100% negative percent agreement for clinical specimens compared with the real-time RT-PCR assay. The new assay is considered useful for diagnosis of rubella in areas where rubella is endemic.

An Immiscible-Phase Filtration Device for Isolation, Amplification, and Detection of Nucleic Acids for Clinical Diagnostics.

Clinical diagnostics of infectious diseases via nucleic acid amplification tests (NAATs) depend on a separate step of isolation of nucleic acids from cells/viruses embedded in complex biological matrices. The most recent example has been reverse transcription polymerase chain reaction (RT-PCR) for amplification and detection of SARS-CoV-2 RNA for COVID-19 diagnostics. Kits for RNA extraction and purification are commercially available; however, their integration with amplification systems is generally lacking, resulting in two separate steps, i.e., sample preparation and amplification. This makes NAATs more time-consuming, requiring skilled personnel, and can increase the likelihood of contamination. Here, we describe a setup and methodology to perform the quick extraction and detection of nucleic acids in an integrated manner. In particular, we focus on the use of an immiscible filtration device for capture, isolation, concentration, amplification, and colorimetric detection of SARS-CoV-2 RNA.

The potential of tailed amplicons for SARS-CoV-2 detection in Nucleic Acid Lateral Flow Assays.

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/µL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9.

BACKGROUND: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. RESULTS: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. CONCLUSIONS: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

Testing for extragenital Neisseria gonorrhoeae and Chlamydia trachomatis: At-home pharyngeal and rectal self-swabs are non-inferior to those completed in healthcare settings.

INTRODUCTION: The rates of gonorrhea and chlamydia have been increasing in the years preceding the COVID19 pandemic. Because most gonorrhea and chlamydia infections are located in the oropharynx and rectum for men who have sex with men (MSM), and because at-home self-collected swabs for these infections are not licensed by Health Canada or the United States Food and Drug Administration, decreased accessed to in-person care during and since the COVID19 pandemic potentially means missed case findings. OBJECTIVES: To evaluate the performance of at-home self-collected pharyngeal and rectal swabs for gonorrhea and chlamydia nucleic acid amplification testing. METHODOLOGY: All persons who contacted our Sexual Health Clinic and who had a clinical indication to complete oral and/or rectal swabs for gonorrhea and chlamydia were invited to complete at-home swabs in advance of their scheduled appointments. We mailed swabs and instructions to those who consented. Participants brought these swabs to their scheduled in clinic appointments, where we repeated the same swabs. All matching swabs were sent to the laboratory for analysis to determine concordance. RESULTS: From September 8, 2022 to July 18, 2023, we enrolled 296 eligible participants who provided 1184 swabs. For analysis, cancelled specimens and specimens with invalid results were excluded, leaving 1032 swabs for comparison. We identified 66 STI diagnoses in 47 unique participants. Overall accuracy was high (exceeding 99%), except for rectal chlamydia, which was 96.0%. While the performance of self-swabs for chlamydia was lower compared to gonorrhea, at-home swabs identified six chlamydia infections that were missed by in-clinic collected swabs (two pharyngeal, four rectal). Removing these six cases as "false positives" increased overall accuracy for chlamydia detection to 99.7% (pharyngeal) and 97.8% (rectal). CONCLUSION: Self-collected at-home swabs had good performance acceptable for gonorrhea and chlamydia nucleic acid amplification testing.

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites.

BACKGROUND: Natural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. METHODS: By using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. RESULTS: There was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. CONCLUSION: This is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer.

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙µL-1, 0.77 ng∙µL-1, and 1.1 ng∙µL-1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙µL-1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.

A Highly Sensitive Molecular Technique for RNA Virus Detection.

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

Spinal Tuberculosis: An Exhaustive Diagnosis.

BACKGROUND: The most common organ affected due to tuberculosis (TB) is the lungs. Extrapulmonary TB is less common. Musculoskeletal organs are affected in around 8% of all tubercular patients, of which the spine is affected in almost half of the patients. The criteria for diagnosing spinal TB are quite difficult and we use an array of investigations for the same. METHODS: A retrospective study was carried out in the Neurosurgery and Microbiology Department at IMS and SUM Hospital between January 2021 and November 2023, and data were collected and tabulated in an Excel sheet. One hundred patients with spinal TB were evaluated, and their age, sex, samples sent, diagnostic investigation, duration of diagnosis from hospital admission, histopathology results, and surgical intervention (done or not) were recorded. RESULTS: The best investigation done to diagnose spinal TB was imaging and surgical/computed tomography (CT)-guided biopsy. The earliest result to diagnose spinal TB was histopathology. The yield of positivity in pus culture, smear microscopy, and true nucleic acid amplification test (NAAT) was found to be low even though sensitivity was on the higher side. CONCLUSION: Even though we have an array of investigations for diagnosing spinal TB, the best and the earliest diagnosing test was imaging plus CT-guided biopsy. The confirmation is made in the biopsy. Finding acid-fast bacteria (AFB) and NAAT tests are additional beneficial tests to supplement the diagnosis. Hence, we can conclude that sending for tests like AFB in pus, NAAT, and GeneXpert is a wastage of biological samples and delays in diagnosis.

Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one.

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, a biosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limit and a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.

Three-dimensional DNA nanomachine biosensor coupled with CRISPR Cas12a cascade amplification for ultrasensitive detection of carcinoembryonic antigen.

The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited accessibility and specificity. This study proposes a rapid and convenient Cas12a-based assay for the direct detection of CEA in clinical serum samples, aiming to address these limitations. The protocol involves a rolling machine operation, followed by a 5-min Cas12a-mediated cleavage process. The assay demonstrates the capability to detect human serum with high anti-interference performance and a detection limit as low as 0.2 ng/mL. The entire testing procedure can be accomplished in 75 min without centrifugation steps, and successfully reduced the limit of detection of traditional DNA walking machine by 50 folds. Overall, the testing procedure can be easily implemented in clinical settings.

Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

Detection and identification of Bogia coconut syndrome phytoplasma from seed-associated tissues and seedlings of coconut (Cocos nucifera) and betel nut (Areca catechu).

Evidence for seed transmission of phytoplasmas has grown in several pathosystems including coconut (Cocos nucifera). Bogia coconut syndrome (BCS) is a disease associated with the lethal yellowing syndrome associated with the presence of 'Candidatus Phytoplasma noviguineense' that affects coconut, betel nut (Areca catechu) and bananas (Musa spp.) in Papua New Guinea. Coconut and betel nut drupes were sampled from BCS-infected areas in Papua New Guinea, dissected, the extracted nucleic acid was used in polymerase chain reaction (PCR), and loop mediated isothermal amplification (LAMP) used to check for presence of phytoplasma DNA. In a second study, drupes of both plant species were collected from multiple field sites and grown in insect-proof cages. Leaf samples taken at 6 months were also tested with PCR and LAMP. The studies of dissected coconut drupes detected phytoplasma DNA in several tissues including the embryo. Drupes from betel nut tested negative. Among the seedlings, evidence of possible seed transmission was found in both plant species. The results demonstrate the presence of 'Ca. P. noviguineense' in coconut drupes and seedlings, and in seedlings of betel nut; factors that need to be considered in ongoing management and containment efforts.

Lateral flow strip assay of a gene segment in the COVID-19 virus with combined dual readout mode and preliminary multisite hybrid chain reaction amplification.

The past and present scenario of COVID-19 has revealed the necessity of simple point-of-care tests. When combined with the great advantages of amplification, lateral flow assay nucleic acid analysis represents a more sensitive molecular diagnostic technique compared to universal protein analysis. Room temperature operation, an enzyme-free nature, and in situ elongation make hybrid chain reaction amplification (HCR) a good candidate for amplified combined lateral flow assays (LFAs). Since dual modes of detection can not only satisfy different application scenarios, but also reduce the false-negative rate, in this paper, visual and fluorescent detection based on labelling with colloidal gold nanoparticles and fluorescence labelling were incorporated into a HCR integrated with a LFA. The detection assay was finished in 30 minutes. The linear relationship between the signal and the concentration of the characteristic segment in the COVID-19 ORF gene was demonstrated. The obtained detection limits of as low as 10 fM (6.02 × 103 copies per mL) and 1 fM (6.02 × 102 copies per mL), respectively, were comparable with those in the literature. The multi-site HCR amplification integrated with LFA of a 1053 bp nucleic acid chain was also preliminarily studied, and tri-site amplification was found to exhibit higher signal intensity than single-site amplification. This study provides a promising strategy for simple, sensitive, and wide-ranging detection of pathogenic bacteria.

A one-pot CRISPR-RCA strategy for ultrasensitive and specific detection of circRNA.

Accurate and precise detection of circular RNA (circRNA) is imperative for its clinical use. However, the inherent challenges in circRNA detection, arising from its low abundance and potential interference from linear isomers, necessitate innovative solutions. In this study, we introduce, for the first time, the application of the CRISPR/Cas12a system to establish a one-pot, rapid (30 minutes to 2 hours), specific and ultrasensitive circRNA detection strategy, termed RETA-CRISPR (reverse transcription-rolling circle amplification (RT-RCA) with the CRISPR/Cas12a). This method comprises two steps: (1) the RT-RCA process of circRNA amplification, generating repeat units containing the back-splicing junction (BSJ) sequences; and (2) leveraging the protospacer adjacent motif (PAM)-independent Cas12a/crRNA complex to precisely recognize target sequences with BSJ, thereby initiating the collateral cleavage activity of Cas12a to generate a robust fluorescence signal. Remarkably, this approach exhibits the capability to detect circRNAs at a concentration as low as 300 aM. The sensor has been successfully employed for accurate detection of a potential hepatocellular carcinoma biomarker hsa_circ_0001445 (circRNA1445) in various cell lines. In conclusion, RETA-CRISPR seamlessly integrates the advantages of exponential amplification reaction and the robust collateral cleavage activity of Cas12a, positioning it as a compelling tool for practical CRISPR-based diagnostics.

Performance of CBNAAT on Pleural Biopsies Obtained by Semirigid Thoracoscopy for Diagnosis of Unexplained Pleural Effusion: A Prospective Study from North India.

BACKGROUND: Exudative pleural effusions are commonly encountered in clinical practice, but in about one-fourth of cases, etiology remains elusive after initial evaluation. Medical thoracoscopy with semirigid thoracoscope is a minimally invasive procedure with high diagnostic yield for diagnosing pleural diseases, especially these undiagnosed exudative pleural effusions. In tubercular endemic areas, often, these effusions turn out to be tubercular, but the diagnosis of tubercular pleural effusion is quite challenging due to the paucibacillary nature of the disease. Although culture is the gold standard, it is time-consuming. Cartridge-based nucleic acid amplification test (CBNAAT) is a novel rapid diagnostic test for tuberculosis (TB) and has been recommended as the initial diagnostic test in patients suspected of having extrapulmonary TB (EPTB). MATERIALS AND METHODS: We conducted a prospective observational study of 50 patients with undiagnosed pleural effusion admitted to our tertiary care hospital. The primary aim of the study is to evaluate the diagnostic performance of CBNAAT on thoracoscopic guided pleural biopsy and compare it with conventional diagnostic techniques like histopathology and conventional culture. RESULTS: Of 50 undiagnosed pleural effusions, TB (50%) was the most common etiology. The overall diagnostic yield of semirigid thoracoscopy in this study was 74%. Our study showed that CBNAAT of pleural biopsies had a sensitivity of 36% only but a specificity of 100%. The sensitivity of CBNAAT was not far superior to the conventional culture. CONCLUSION: Tuberculosis (TB) is a common cause of undiagnosed pleural effusion in our set-up. CBNAAT testing of pleural biopsy, though, is a poor rule-out test for pleural TB, but it may aid in the early diagnosis of such patients.

Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19.

Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

A LAMP-based hydrogen ion selective electrochemical sensor for highly sensitive detection of Mycoplasma pneumoniae.

A concise and rapid detection method for Mycoplasma pneumoniae is urgently required due to its severe impact on human health. To meet such a need, this study proposed and constructed an innovative point-of-care testing (POCT) platform that consists of a hydrogen ion-selective loop-mediated isothermal amplification (H+-LAMP) sensor and an electrochemical detection device. The H+-LAMP sensor successfully integrated the working and reference electrodes and converted the H+ generated during the LAMP process into an electrochemical signal. High sensitivity and stability for pathogen detection were also achieved by treating the working electrode with an electrodeposited polyaniline solid contact layer and by using an ion-selective membrane. As a result, the sensor shows a sensitivity of 68.26 mV per pH, a response time of less than 2 s, and a potential drift of less than 5 mV within one hour, which well meets the urgent need. The results also demonstrated that the detection limit for Mycoplasma pneumoniae was lowered to 1 copy per µL, the nucleic acid extraction and detection process could be completed in 30 minutes, and the impact of interfering ions on the sensor was negligible. Validation with 20 clinical samples yielded satisfactory results. More importantly, the storage lifespan of such an electrochemical sensor is over seven days, which is a great advantage for on-site pathogen detection. Therefore, the hydrogen ion-selective sensor constructed in this investigation is particularly suitable as a core component for instant pathogen detection platforms.