Biosensor-based multiple cross displacement amplification platform for visual and rapid identification of hepatitis C virus.

Hepatitis C remains a global health problem, especially in poverty-stricken areas. A rapid and sensitive point-of-care (POC) diagnostic tool is critical for the early detection and timely treatment of hepatitis C virus (HCV) infection. Here, for the first time, we reported a novel molecular diagnostic assay, termed reverse transcription multiple cross displacement amplification integrated with a gold-nanoparticle-based lateral flow biosensor (RT-MCDA-AuNPs-LFB), which was developed for rapid, sensitive, specific, and visual identification of HCV. HCV-RT-MCDA induced rapid isothermal amplification through a specific primer set targeting the 5'untranslated region gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a that are prevalent in China. The optimal reaction temperature and time for RT-MCDA-AuNPs-LFB were 68°C and 25 min, respectively. The limit of detection of the assay was 10 copies per test, and the specificity was 100% for the experimental strains. The whole detection procedure, including crude nucleic acid isolation (~5 min), RT-MCDA (68°C, 25 min), and visual AuNPs-LFB result confirmation (less than 2 min), was performed within 35 min. The preliminary results indicated that the HCV-RT-MCDA-AuNPs-LFB assay could be a valuable tool for sensitive, specific, visual, cost-saving, and rapid detection of HCV and has potential as a POC diagnostic platform for field screening and early clinical detection of HCV infection.

Relationship between vitamin D levels and pediatric celiac disease: a systematic review and meta-analysis.

BACKGROUND: The relationship between Vitamin D levels and pediatric celiac disease (CD) remains controversial. In this study, we conducted a systematic review and meta-analysis to examine the relationship between Vitamin D and pediatric CD. METHODS: We screened relevant studies from PubMed, EMBASE, and Web of Science published in English from January 1, 2000, to August 1, 2023. The included studies were assessed according to the STROBE checklist. Heterogeneity was quantified by Cochran's Q test and the I2 statistic. Publication bias was estimated by Begg's test and Egger's test. Meta-regression was used to detect potential sources of heterogeneity. RESULTS: A total of 26 studies were included in the meta-analysis. Nineteen articles compared 25(OH)D3 levels between CD patients and control groups, average 25-hydroxyvitamin D3 [25(OH)D3 or calcidiol], and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3 or calcitriol] levels, as the main forms of Vitamin D, there was a significant difference in CD patients and healthy controls (weighted mean difference (WMD) = - 5.77, 95% confidence interval (CI) = [- 10.86, - 0.69] nmol/L). Meanwhile, eleven articles reported the numbers of patients and controls with Vitamin D deficiency, there was a significant difference in the incidence of 25(OH)D3 deficiency between CD patients and healthy controls (odds ratio 2.20, 95% CI= [1.19, 4.08]). Nine articles reported changes in 25(OH)D3 levels before and after administering a GFD in patients with CD, the result of this study revealed the increase of 25(OH)D3 levels in CD patients after a gluten-free diet (GFD) (WMD = - 6.74, 95% CI = [- 9.78, - 3.70] nmol/L). CONCLUSIONS: Vitamin D levels in pediatric CD patients were lower than in healthy controls, and 25(OH)D3 deficiency was more prevalent in CD patients. We found that 25(OH)D3 levels were elevated in CD patients after GFD, which is consistent with previous research. Further well-designed, longitudinal, prospective cohort studies focusing on the role of Vitamin D in the pathogenesis of CD are therefore needed.

Rapid, visual, label-based biosensor platform for identification of hepatitis C virus in clinical applications.

OBJECTIVES: In the current study, for the first time, we reported a novel HCV molecular diagnostic approach termed reverse transcription loop-mediated isothermal amplification integrated with a gold nanoparticles-based lateral flow biosensor (RT-LAMP-AuNPs-LFB), which we developed for rapid, sensitive, specific, simple, and visual identification of HCV. METHODS: A set of LAMP primer was designed according to 5'untranslated region (5'UTR) gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a, which are prevalent in China. The HCV-RT-LAMP-AuNPs-LFB assay conditions, including HCV-RT-LAMP reaction temperature and time were optimized. The sensitivity, specificity, and selectivity of our assay were evaluated in the current study. The feasibility of HCV-RT-LAMP-AuNPs-LFB was confirmed through clinical serum samples from patients with suspected HCV infections. RESULTS: An unique set of HCV-RT-LAMP primers were successfully designed targeting on the 5'UTR gene. The optimal detection process, including crude nucleic acid extraction (approximately 5 min), RT-LAMP reaction (67℃, 30 min), and visual interpretation of AuNPs-LFB results (~ 2 min), could be performed within 40 min without specific instruments. The limit of detection was determined to be 20 copies per test. The HCV-RT-LAMP-AuNPs-LFB assay exhibited high specificity and anti-interference. CONCLUSIONS: These preliminary results confirmed that the HCV-RT-LAMP-AuNPs-LFB assay is a sensitive, specific, rapid, visual, and cost-saving assay for identification of HCV. This diagnostic approach has great potential value for point-of-care (POC) diagnostic of HCV, especially in resource-challenged regions.

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

Sexually transmitted chlamydia and gonorrhea infections caused by the bacteria Chlamydia trachomatis and Neisseria gonorrhoeae remain a major public health concern worldwide, particularly in less developed nations. It is crucial to use a point of care (POC) diagnostic method that is quick, specific, sensitive, and user-friendly to treat and control these infections effectively. Here, a novel molecular diagnostic assay, combining multiplex loop-mediated isothermal amplification (mLAMP) with a visual gold nanoparticles-based lateral flow biosensor (AuNPs-LFB) was devised and used for highly specific, sensitive, rapid, visual, and easy identification of C. trachomatis and N. gonorrhoeae. Two unique independent primer pairs were successful designed against the ompA and orf1 genes of C. trachomatis and N. gonorrhoeae, respectively. The optimal mLAMP-AuNPs-LFB reaction conditions were determined to be 67°C for 35 min. The detection procedure, involving crude genomic DNA extraction (~5 min), LAMP amplification (35 min), and visual results interpretation (<2 min), can be completed within 45 min. Our assay has a detection limit of 50 copies per test, and we did not observe any cross-reactivity with any other bacteria in our testing. Hence, our mLAMP-AuNPs-LFB assay can potentially be used for POC testing to detect C. trachomatis and N. gonorrhoeae in clinical settings, particularly in underdeveloped regions.

Sensitive and visual identification of Chlamydia trachomatis using multiple cross displacement amplification integrated with a gold nanoparticle-based lateral flow biosensor for point-of-care use.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection (STI) and remains a major public health challenge, especially in less-developed regions. Establishing a rapid, inexpensive, and easy-to-interpret point-of-care (POC) testing system for C. trachomatis could be critical for its treatment and limiting further transmission. Here, we devised a novel approach termed a multiple cross displacement amplification integrated with gold nanoparticle-based lateral flow biosensor (MCDA-AuNPs-LFB) for the highly specific, sensitive, user-friendly, and rapid identification of C. trachomatis in clinical samples. A suite of MCDA primers based on the C. trachomatis ompA gene from 14 serological variants (serovar A-K, L1, L2, and L3) were successfully designed and used to establish the assay. Optimal assay conditions were identified at 67°C, and the detection procedure, including nucleic acid preparation (approximately 5 min), MCDA amplification (30 min), and AuNPs-LFB visual readout (within 2 min), was completed within 40 min. The all-in cost for each test was approximately $5.5 USD. The limit of detection (LoD) was 10 copies/reaction, and no cross-reaction was observed with non-C. trachomatis microbes. A total of 135 suspected C. trachomatis-infection genital secretion samples were collected and simultaneously detected using real-time quantitative PCR (qPCR) in our assay. Compared with the qPCR technology, the MCDA-AuNPs-LFB sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 96.20%, 94.92%, and 100%, respectively. Hence, our MCDA-AuNP-LFB assay exhibited considerable potential for POC testing and could be used to identify C. trachomatis in clinical settings, particularly in low-income 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.

Chlamydial infection, caused by Chlamydia trachomatis, is the most common bacterial sexually transmitted infection and remains a major public health problem worldwide, particularly in underdeveloped regions. Developing a rapid and sensitive point-of-care (POC) testing for accurate screening of C. trachomatis infection is critical for earlier treatment to prevent transmission. In this study, a novel diagnostic assay, loop-mediated isothermal amplification integrated with gold nanoparticle-based lateral flow biosensor (LAMP-LFB), was devised and applied for diagnosis of C. trachomatis in clinical samples. A set of LAMP primers based on the ompA gene from 14 C. trachomatis serological variants (serovar A-K, L1, L2, L3) was successfully designed and used for the development of C. trachomatis-LAMP-LFB assay. The optimal reaction system can be performed at a constant temperature of 67°C for 35 min. The total assay process, including genomic DNA extraction (~15 min), LAMP reaction (35 min), and LFB readout (~2 min), could be finished within 60 min. The C. trachomatis-LAMP-LFB could detect down to 50 copies/ml, and the specificity was 100%, no cross-reactions with other pathogens were observed. Hence, our C. trachomatis-LAMP-LFB was a rapid, reliable, sensitive, cost-effective, and easy-to-operate assay, which could offer an attractive POC testing tool for chlamydial infection screening, especially in resource starvation settings.

A Nanoparticle-Based Biosensor Combined With Multiple Cross Displacement Amplification for the Rapid and Visual Diagnosis of Neisseria gonorrhoeae in Clinical Application.

Gonorrhea is a sexually transmitted disease caused by the host-adapted human pathogen, Neisseria gonorrhoeae. The morbidity is increasing and poses a major public health concern, especially in resource-scarce regions. Therefore, a rapid, visual, sensitive, specific, cost-saving, and simple assay for N. gonorrhoeae detection is critical for prompt treatment and the prevention of further transmission. Here, for the first time, we report a novel assay called the multiple cross displacement amplification combined with gold nanoparticle-based lateral flow biosensor (MCDA-LFB), which we constructed for the rapid and visual identification of N. gonorrhoeae in clinical samples. We successfully devised a set of MCDA primers based on the N. gonorrhoeae-specific gene, orf1. Optimal assay conditions were determined at 67°C, including genomic DNA preparation (∼15 min), MCDA amplification (30 min), and LFB reading (∼2 min), which can be completed within 50 min. The limit of detection (LoD) of the assay was 20 copies/test (in a 25-µl reaction mixture). Assay specificity was 100%, with no cross-reactions with other pathogens. Thus, our N. gonorrhoeae-MCDA-LFB is a rapid, specific, visual, cost-saving, and easy-to-use assay for N. gonorrhoeae diagnostics, and may have great potential for point-of-care (POC) testing in clinical settings, especially in resource-limited regions.

Protein degradation control and regulation of bacterial survival and pathogenicity: the role of protein degradation systems in bacteria.

BACKGROUND: Protein degradation systems play crucial roles in all the kingdoms of life. Their natural function is to eliminate proteins that are improperly synthesized, damaged, aggregated, or short-lived, ensuring the timely and accurate regulation of the response to abrupt environmental changes. Thus, proteolysis plays an important role in protein homeostasis, quality control, and the control of regulatory processes, such as adaptation and cell development. Except for the lysosome, ATPases Associated with various cellular Activities (AAA+) ATPase-protease complex is another major protein degradation system in the cell. METHODS AND RESULTS: The AAA+ ATPase-protease complex is a giant energy-dependent protease complex found in almost all kinds of cells, including bacteria, archaea and eukarya. Based on sequence analysis of ClpQ (HslV) and 20S proteasome beta subunits, it was found that bacterial ClpQ possess multiple same highly conserved motifs with 20S proteasome beta subunits of archaea and eukaryote. In this review, we also discussed the structure and functional mechanism, protein degradation signals and pathogenic role of proteasome / Clp protease complex in prokaryotes. CONCLUSION: Bacterial protein degradation systems play important roles in stress tolerance, protein quality control, DNA protection, transcription and pathogenicity of bacteria. But our current knowledge of the bacterial protease system is incomplete, and further research into the Clp protease complex and associated protein degradation signals will extend our understanding of the metabolism, physiology, reproduction, and pathogenicity of bacteria.

Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor.

Background: Severe 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. Methods: A 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. Results: The 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. Conclusion: The 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.

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

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.

Multiple Cross Displacement Amplification Linked with Nanoparticles-Based Lateral Flow Biosensor in Screening of Hepatitis B Virus in Clinical Application.

BACKGROUND: Hepatitis B virus (HBV) is a common pathogen that predominantly causes severe liver disease, and remains one of a huge challenge worldwide, especially in many resource-constrained areas. Developing a low-cost, sensitive, specific, and rapid approach for screening HBV is critical for its treatment and prevention. In the current study, a novel molecular detection approach, multiple cross displacement amplification (MCDA) coupled with polymer nanoparticle-based lateral flow biosensor (MCDA-LFB), was applied for detection of HBV in blood samples. METHODS: HBV standard substance and clinical donor serum samples were collected and used for the establishment and confirmation of the HBV-MCDA-LFB assay. A set of 10 MCDA primers was designed according to HBV-specific gene S. The HBV-MCDA-LFB assay conditions, including genomic template concentration, MCDA reaction temperature and time were optimized. The sensitivity and specificity of the HBV-MCDA -LFB assay were evaluated in this report. The HBV-MCDA-LFB assay was applied to detect the HBV agent from clinical samples. RESULTS: The HBV-MCDA primers based on the S gene were valid for establishment of MCDA assay. The HBV-MCDA reaction with optimized conditions could be carried out at a constant temperature 64°C for 35 min. The whole process, including sample preparation (5 min), genomic template extraction (~30 min), MCDA amplification (35 min), and LFB reading (~2 min), could be completed within 80 min. The sensitivity of this assay was 5 IU per reaction. The specificity was 100% for HBV-MCDA-LFB assay. CONCLUSION: These results confirmed that the HBV-MCDA-LFB is a low-cost, sensitive, specific, simple, and rapid method for detecting HBV agents. This technique has great potential to develop a point-of-care testing (POCT) method in clinical practice, especially in endemic and resource-constrained regions.

Screening of Concentration and Antimicrobial Effectiveness of Antimicrobial Preservative in Betastatin Besylate Nasal Spray.

OBJECTIVE: To explore the optimal concentration and antimicrobial effectiveness of antimicrobial preservative in betastatin besylate nasal spray. METHODS: By using Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Aspergillus niger as test strains, the antimicrobial effectiveness of betastatin besylate nasal spray containing different concentrations of antimicrobial preservative (0.02%, 0.0125%, and 0.005% benzalkonium chloride, respectively) was determined by using bacteriostatic effect test (Chinese Pharmacopoeia, 2015 edition). RESULTS: The antimicrobial effectiveness of betastatin besylate nasal spray containing 0.02% and 0.0125% benzalkonium chloride, respectively, complied with the regulations of Chinese Pharmacopoeia (2015 Edition) against five test strains. However, the antimicrobial effectiveness of betastatin besylate nasal spray containing 0.005% benzalkonium chloride against P. aeruginosa did not meet the requirements of Chinese Pharmacopoeia. CONCLUSION: Benzalkonium chloride at a concentration of 0.125% can be used as an added antimicrobial preservative in betastatin besylate nasal spray.