The COVID-19 pandemic evidenced the urgent need for rapid,accurate,and scalable diagnostic methods for emerging infectious diseases.Droplet digital reverse transcription LAMP(ddRT-LAMP)is a promising technique for pat...The COVID-19 pandemic evidenced the urgent need for rapid,accurate,and scalable diagnostic methods for emerging infectious diseases.Droplet digital reverse transcription LAMP(ddRT-LAMP)is a promising technique for pathogen detection and accurate quantification,as it overcomes traditional LAMP’s limitations in viral load estimation through reaction partitioning and digital analysis.However,many parameters must be adjusted to avoid spurious results.This study evaluates the critical conditions for effective ddRT-LAMP quantification of the SARS-CoV-2 N gene in plasmid DNA,synthetic RNA,and nasopharyngeal swab samples.Using a polydimethylsiloxane(PDMS)microfluidic device,the RT-LAMP reaction mixture with a fluorescent dye was divided into thousands of droplets stabilized by a surfactant in fluorinated oil.After incubation,the droplets were injected into a PDMS chamber for fluorescent imaging to determine the proportion of positive droplets and quantify the samples based on the Poisson distribution.The results showed that primer design and master mix composition significantly impacted the amplification.The selection of GelGreen^(R)as the fluorescent dye was crucial,as other dyes tested diffused into the oil phase.Optimal amplification occurred with 105μm droplet diameter and 30-min incubation,achieving detection and quantification limits of 102 cp/μL.By addressing these operational challenges,ddRT-LAMP can become a more effective tool for viral detection and quantification in clinical diagnostics.展开更多
Background Schistosomiasis,caused by parasitic flatworms of the genus Schistosoma,remains a significant public health challenge in tropical and subtropical regions,affecting over hundreds of millions of people in thes...Background Schistosomiasis,caused by parasitic flatworms of the genus Schistosoma,remains a significant public health challenge in tropical and subtropical regions,affecting over hundreds of millions of people in these areas.Accurate diagnosis is crucial for effective disease control,particularly in low-endemic areas where traditional methods like microscopy are no longer effective.We aimed to evaluate the diagnostic performance of loop-mediated isothermal amplification(LAMP)for Schistosoma infection.Methods Adhering to Preferred reporting items for systematic reviews and meta-analyses guidelines,we conducted a comprehensive search on 10 May 2025 across multiple databases including PubMed,Cochrane Library,Latin American and Caribbean Literature on Health Sciences,Embase,China National Knowledge Infrastructure,and Wanfang Data,using keywords such as"schistosom*","LAMP",and"loop-mediated isothermal amplification".Based on available literature,pooled sensitivity,specificity,positive likelihood ratio(PLR),negative likelihood ratio(NLR)and 95%confidential interval(CI)were calculated using STATA18.0 software.Subgroup analyses and univariable meta-regression were performed to explore the source of heterogeneity.Specifically,subgroup analyses were performed by categorizing into species(S.japonicum,S.mansoni,S.haematobium),sample type(stool,urine,serum,snails),and DNA extraction methods to explore factors influencing test performance.Results The study finally included 24 individual studies derived from 14 published articles.The pooled analyses of LAMP data from all included studies resulted in a sensitivity of 0.90(95%CI:0.80-0.90),specificity of 0.82(95%CI:0.60-0.93),PLR of 4.98(95%CI:2.01-12.29),NLR of 0.13(95%CI:0.06-0.26)and diagnostic odds ratio of 39(95%CI:10-158).The area under the summary receiver operating characteristic curve reached 0.93,indicating excellent diagnostic performance.Subgroup analyses revealed optimal performance for S.japonicum and snail samples with lower heterogeneity(I2<50%).Conclusions LAMP shows promise as a rapid,sensitive and specific diagnostic tool for schistosomiasis,particularly in resource-limited settings.This technique enables field application,supporting global efforts toward elimination of schistosomiasis by 2030.展开更多
Listeriosis is caused by Listeria monocytogenes(LM) and is currently considered to be one of the leading food-borne diseases worldwide, with mortality rate of 20%~30%. Currently, detection methods for LM are time-cons...Listeriosis is caused by Listeria monocytogenes(LM) and is currently considered to be one of the leading food-borne diseases worldwide, with mortality rate of 20%~30%. Currently, detection methods for LM are time-consuming with low sensitivity, and delayed detection results. SYTO9 has a high affinity for DNA and exhibits enhanced fluorescence upon binding. Therefore, this study used SYTO9 staining and image processing to develop a rapid loop mediated isothermal amplification(LAMP) detection method for LM. Smartphone was successfully used for detecting the color change in different concentrations of LM. Besides, the optimized LAMP reaction temperature was 63 °C by color identification, and the limit of detection for LM was 6 copies/μL in the green channel. So, the developed method, based on image processing, is simple, sensitive and rapid, which provides a new idea and method for rapid detection of LM and other food-borne bacterial pathogens.展开更多
基金preliminary LAMP experiments in bulk by Dr.Elizabeth Castillo-Villanueva.K.C.R.acknowledges Secretaría de Ciencia,Humanidades,Tecnología e Innovación(SECIHTI),for a postdoctoral scholarship(CVU 662463)supported by UNAM-PAPIIT grants IT100922 and IN211623,SECTEI grant SECTEI/248/2021the Faculty of Chemistry,UNAM(PAIP 5000-9023).
文摘The COVID-19 pandemic evidenced the urgent need for rapid,accurate,and scalable diagnostic methods for emerging infectious diseases.Droplet digital reverse transcription LAMP(ddRT-LAMP)is a promising technique for pathogen detection and accurate quantification,as it overcomes traditional LAMP’s limitations in viral load estimation through reaction partitioning and digital analysis.However,many parameters must be adjusted to avoid spurious results.This study evaluates the critical conditions for effective ddRT-LAMP quantification of the SARS-CoV-2 N gene in plasmid DNA,synthetic RNA,and nasopharyngeal swab samples.Using a polydimethylsiloxane(PDMS)microfluidic device,the RT-LAMP reaction mixture with a fluorescent dye was divided into thousands of droplets stabilized by a surfactant in fluorinated oil.After incubation,the droplets were injected into a PDMS chamber for fluorescent imaging to determine the proportion of positive droplets and quantify the samples based on the Poisson distribution.The results showed that primer design and master mix composition significantly impacted the amplification.The selection of GelGreen^(R)as the fluorescent dye was crucial,as other dyes tested diffused into the oil phase.Optimal amplification occurred with 105μm droplet diameter and 30-min incubation,achieving detection and quantification limits of 102 cp/μL.By addressing these operational challenges,ddRT-LAMP can become a more effective tool for viral detection and quantification in clinical diagnostics.
基金supported by the Key Discipline Project(GWVI-11.1-12)of the Three-Year Action Plan for the Construction of Shanghai Public Health System(2023-2025).
文摘Background Schistosomiasis,caused by parasitic flatworms of the genus Schistosoma,remains a significant public health challenge in tropical and subtropical regions,affecting over hundreds of millions of people in these areas.Accurate diagnosis is crucial for effective disease control,particularly in low-endemic areas where traditional methods like microscopy are no longer effective.We aimed to evaluate the diagnostic performance of loop-mediated isothermal amplification(LAMP)for Schistosoma infection.Methods Adhering to Preferred reporting items for systematic reviews and meta-analyses guidelines,we conducted a comprehensive search on 10 May 2025 across multiple databases including PubMed,Cochrane Library,Latin American and Caribbean Literature on Health Sciences,Embase,China National Knowledge Infrastructure,and Wanfang Data,using keywords such as"schistosom*","LAMP",and"loop-mediated isothermal amplification".Based on available literature,pooled sensitivity,specificity,positive likelihood ratio(PLR),negative likelihood ratio(NLR)and 95%confidential interval(CI)were calculated using STATA18.0 software.Subgroup analyses and univariable meta-regression were performed to explore the source of heterogeneity.Specifically,subgroup analyses were performed by categorizing into species(S.japonicum,S.mansoni,S.haematobium),sample type(stool,urine,serum,snails),and DNA extraction methods to explore factors influencing test performance.Results The study finally included 24 individual studies derived from 14 published articles.The pooled analyses of LAMP data from all included studies resulted in a sensitivity of 0.90(95%CI:0.80-0.90),specificity of 0.82(95%CI:0.60-0.93),PLR of 4.98(95%CI:2.01-12.29),NLR of 0.13(95%CI:0.06-0.26)and diagnostic odds ratio of 39(95%CI:10-158).The area under the summary receiver operating characteristic curve reached 0.93,indicating excellent diagnostic performance.Subgroup analyses revealed optimal performance for S.japonicum and snail samples with lower heterogeneity(I2<50%).Conclusions LAMP shows promise as a rapid,sensitive and specific diagnostic tool for schistosomiasis,particularly in resource-limited settings.This technique enables field application,supporting global efforts toward elimination of schistosomiasis by 2030.
基金supported by the grant from the National Natural Science Foundation of China (Nos. 61901168, 8200240581902153)+1 种基金Zhuzhou Innovative City Construction Project(No. 2020–020)China Postdoctoral Science Foundation (No.2018M630498)。
文摘Listeriosis is caused by Listeria monocytogenes(LM) and is currently considered to be one of the leading food-borne diseases worldwide, with mortality rate of 20%~30%. Currently, detection methods for LM are time-consuming with low sensitivity, and delayed detection results. SYTO9 has a high affinity for DNA and exhibits enhanced fluorescence upon binding. Therefore, this study used SYTO9 staining and image processing to develop a rapid loop mediated isothermal amplification(LAMP) detection method for LM. Smartphone was successfully used for detecting the color change in different concentrations of LM. Besides, the optimized LAMP reaction temperature was 63 °C by color identification, and the limit of detection for LM was 6 copies/μL in the green channel. So, the developed method, based on image processing, is simple, sensitive and rapid, which provides a new idea and method for rapid detection of LM and other food-borne bacterial pathogens.
