Objective: The objective of the study is to verify the clinical validity of the following kits with the comparative experimental analysis and evaluate whether their performance can meet the clinical requirements, i.e....Objective: The objective of the study is to verify the clinical validity of the following kits with the comparative experimental analysis and evaluate whether their performance can meet the clinical requirements, i.e. Class III in vitro diagnostic reagent “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (PCR-Fluorescence Probe Method)” of Daan Gene Co., Ltd. (Daan kit for short) and “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (Fluorescence PCR Method)” of Wuhan Biot Gene Co., Ltd. (Biot kit for short). Method: In the study process, the samples were divided into positive and negative groups according to the control test results, and the clinical application performance of Daan kit and Biot kit was evaluated by comparing their test results. Results: The results show that two kits indicate the same test results, i.e. 26 positive and 107 negative samples in a total of 133 male urethral discharge samples, and 32 positive and 238 negative samples in a total of 270 female cervical secretion samples. Conclusion: It can be concluded from the clinical test that Daan and Biot Herpes Simplex Virus (HSV) Type II Nuc- leic Acid Test Kits are reliable, accurate, safe, convenient for use, stable and high-value in the clinical application.展开更多
The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop...The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.展开更多
Nucleic acid detection plays a key role in diverse diagnosis and disease control.Currently available nucleic acid detection techniques are challenged by trade-offs among speed,simplicity,precision and cost.Here,we des...Nucleic acid detection plays a key role in diverse diagnosis and disease control.Currently available nucleic acid detection techniques are challenged by trade-offs among speed,simplicity,precision and cost.Here,we described a novel method,designated SENSOR(Sulfur DNA mediated nucleic acid sensing platform),for rapid nucleic acid detection.SENSOR was developed from phosphorothioate(PT)-DNA and sulfur binding domain(SBD)which specifically binds double-stranded PT-modified DNA.SENSOR utilizes PT-DNA oligo and SBD as targeting module,which is linked with split luciferase reporter to generate luminescence signal within 10 min.We tested detection on synthesized nucleic acid and COVID-19 pseudovirus,achieving attomolar sensitivity combined with an amplification procedure.Single nucleotide polymorphisms(SNP)could also be discriminated.Indicating SENSOR a new promising nucleic acid detection technique.展开更多
Schistosomiasis is an important zoonotic parasitic disease that causes serious harms to humans and animals.Surveillance and diagnosis play key roles in schistosomiasis control,however,current techniques for surveillan...Schistosomiasis is an important zoonotic parasitic disease that causes serious harms to humans and animals.Surveillance and diagnosis play key roles in schistosomiasis control,however,current techniques for surveillance and diagnosis of the disease have limitations.As genome data for parasites are increasing,novel techniques for detection incorporating nucleotide sequences are receiving widespread attention.These sensitive,specific,and rapid detection methods are particularly important in the diagnosis of low-grade and early infections,and may prove to have clinical significance.This paper reviews the progress of nucleic acid detection in the diagnosis and prevention of schistosomiasis,including such aspects as the selection of target genes,and development and application of nucleic acid detection methods.展开更多
Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to pre...Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to prevent the further spread of the epidemic. An optimized RPA-Cas12a-based platform combined with digital microfluidics(DMF), the RCD platform, was established to achieve the automated, rapid detection of influenza viruses and SARS-CoV-2. The probe in the RPA-Cas12a system was optimized to produce maximal fluorescence to increase the amplification signal. The reaction droplets in the platform were all at the microliter level and the detection could be accomplished within 30 min due to the effective mixing of droplets by digital microfluidic technology. The whole process from amplification to recognition is completed in the chip, which reduces the risk of aerosol contamination. One chip can contain multiple detection reaction areas, offering the potential for customized detection.The RCD platform demonstrated a high level of sensitivity, specificity(no false positives or negatives), speed(≤30 min),automation and multiplexing. We also used the RCD platform to detect nucleic acids from influenza patients and COVID-19 patients. The results were consistent with the findings of q PCR. The RCD platform is a one-step, rapid, highly sensitive and specific method with the advantages of digital microfluidic technology, which circumvents the shortcomings of manual operation. The development of the RCD platform provides potential for the isothermal automatic detection of nucleic acids during epidemics.展开更多
BACKGROUND The global outbreak of human severe acute respiratory syndrome coronavirus(SARS-CoV)-2 infection represents an urgent need for readily available,accurate and rapid diagnostic tests.Nucleic acid testing of r...BACKGROUND The global outbreak of human severe acute respiratory syndrome coronavirus(SARS-CoV)-2 infection represents an urgent need for readily available,accurate and rapid diagnostic tests.Nucleic acid testing of respiratory tract specimens for SARS-CoV-2 is the current gold standard for diagnosis of coronavirus disease 2019(COVID-19).However,the diagnostic accuracy of reverse transcription polymerase chain reaction(RT-PCR)tests for detecting SARS-CoV-2 nucleic acid may be lower than optimal.The detection of SARS-CoV-2-specific antibodies should be used as a serological non-invasive tool for the diagnosis and management of SARS-CoV-2 infection.AIM To investigate the diagnostic value of SARS-CoV-2 IgM/IgG and nucleic acid detection in COVID-19.METHODS We retrospectively analyzed 652 suspected COVID-19 patients,and 206 non-COVID-19 patients in Wuhan Integrated TCM and Western Medicine Hospital.Data on SARS-CoV-2 nucleic acid tests and serum antibody tests were collected to investigate the diagnostic value of nucleic acid RT-PCR test kits and immunoglobulin(Ig)M/IgG antibody test kits.The j2 test was used to compare differences between categorical variables.A 95%confidence interval(CI)was provided by the Wilson score method.All analyses were performed with IBM SPSS Statistics version 22.0(IBM Corp.,Armonk,NY,United States).RESULTS Of the 652 suspected COVID-19 patients,237(36.3%)had positive nucleic acid tests,311(47.7%)were positive for IgM,and 592(90.8%)were positive for IgG.There was a significant difference in the positive detection rate between the IgM and IgG test groups(P<0.001).Using the RT-PCR results as a reference,the specificity,sensitivity,and accuracy of IgM/IgG combined tests for SARS-CoV-2 infection were 98.5%,95.8%,and 97.1%,respectively.Of the 415 suspected COVID-19 patients with negative nucleic acid test results,366 had positive IgM/IgG tests with a positive detection rate of 88.2%.CONCLUSION Our data indicate that serological IgM/IgG antibody combined test had high sensitivity and specificity for the diagnosis of SARS-CoV-2 infection,and can be used in combination with RT-PCR for the diagnosis of SARS-CoV-2 infection.展开更多
Nucleic acid detection,widely used in clinical diagnosis,biological analysis,and environmental monitoring,is of great significance for disease diagnosis and basic research.With the outbreak of COVID-19,the demand for ...Nucleic acid detection,widely used in clinical diagnosis,biological analysis,and environmental monitoring,is of great significance for disease diagnosis and basic research.With the outbreak of COVID-19,the demand for fast and high-throughput nucleic acid detection from large numbers of samples has increased sharply.Automated nucleic acid detection systems can meet these needs,and also play important roles in disease screening and infectious disease prevention and control.In this review,we introduce and compare the current mainstream nucleic acid automatic detection instruments and equipment,then discuss the future demands of nucleic acid detection.展开更多
Nucleic acid detection(NAD)based on real-time polymerase chain reaction(real-time PCR)is gold standard for infectious disease detection.Magnetic nanoparticles(MNPs)are widely used for nucleic acid extraction(NAE)becau...Nucleic acid detection(NAD)based on real-time polymerase chain reaction(real-time PCR)is gold standard for infectious disease detection.Magnetic nanoparticles(MNPs)are widely used for nucleic acid extraction(NAE)because of their excellent properties.Microfluidic technology makes automated NAD possible.However,most of the NAD microfluidic chips are too complex to be applied to point-of-care(POC)testing.In this paper,a simple-structure cartridge was developed for POC detection of infectious diseases.This self-contained cartridge can be divided into a magnetic-controlled NAE part,a valve-piston combined fluidic control part and a PCR chip,which is able to extract nucleic acid from up to 500μL of liquid samples by MNPs and finish the detection process from“sample in”to“answer out”automatically.Performance tests of the cartridges show that it met the demands of automated NAD.Results of on-cartridge detection of hepatitis B virus(HBV)demonstrated that this system has good uniformity and no cross-contamination between different cartridges,and the limit of detection(LOD)of this system for HBV in serum is 50 IU/mL.Multiplex detections of severe acute respiratory syndrome coronaviruses 2(SARS-CoV-2)with a concentration of 500 copies/mL were carried out on the system and 100%positive detection rate was achieved.展开更多
Coronavirus disease 2019(COVID-19)has been a pandemic for more than a year.With the expanding second wave of the pandemic in winter,the continuous evolution of SARS-CoV-2 has brought new issues,including the significa...Coronavirus disease 2019(COVID-19)has been a pandemic for more than a year.With the expanding second wave of the pandemic in winter,the continuous evolution of SARS-CoV-2 has brought new issues,including the significance of virus mutations in infection and the detection of asymptomatic infection.In this review,we first introduced several major SARS-CoV-2 mutations since the COVID-19 outbreak and then mentioned the widely used molecular detection techniques to diagnose COVID-19,primarily focusing on their strengths and limitations.We further discussed the effects of viral genetic variation and asymptomatic infection on the molecular detection of SARS-CoV-2 infection.The review finally summarized useful insights into the molecular diagnosis of COVID-19 under the special situation being challenged by virus mutation and asymptomatic infection.展开更多
Objective: The study aims to understand the characteristics and epidemic trend of the pathogen of hand, foot and mouth disease (HFMD) in Guangxi regions, China. Besides, it aims to analyze the differences of intestina...Objective: The study aims to understand the characteristics and epidemic trend of the pathogen of hand, foot and mouth disease (HFMD) in Guangxi regions, China. Besides, it aims to analyze the differences of intestinal virus detection rate between anal swab and pharyngeal swab samples. Methods: Anal swab and pharyngeal swabs of suspected HFMD children were collected in our hospital from 2012 to 2015. Real-time fluorescent PCR (Polymerase Chain Reaction) was used to detect enterovirus 71 (EV71), coxsackie virus type 16 (CA16), and universal intestinal virus nucleic acid (EV). Composition and conversion of predominant pathogens were analyzed, and paired samples’ test results of swabs anal and pharyngeal swab were statistically analyzed. Results: There are 681 cases with enterovirus in 2351 cases of patients. Among those who got enterovirus, there are 501 cases of EV71, 102 cases of CA16 and 79 cases of EV. From 2012 to 2015, the total proportion of the virus detection is 46.47%, 16.23%, 41.02% and 15.33% respectively in each year, while the proportion of predominant epidemic virus is 93.93% of EV71, 66.12% of CA16, 89.30% of EV71 and 98.73% of EV, non-EV71, non-CA16 EV (from October to December in 2015). It’s obvious that the total virus detection rate in 2012 and 2014 is significantly higher than that in 2013 and 2015. There is statistical significance. Conclusion: The main HFMD pathogens are EV71 from 2012 to 2015 in Guangxi regions. In 2012 and 2014, the predominant epidemic pathogens were EV71, while in 2013 and 2015, the predominant epidemic pathogens turn to be CA16 and non-EV71, non-CA16 EV respectively. What’s more, collecting anal swab and pharyngeal swab virus at the same time for nucleic acid detection is of great significance to improve the HFMD laboratory diagnostic.展开更多
The COVID-19 pandemic has brought great challenges to traditional nucleic acid detection technology.Thus,it is urgent to develop a more simple and efficient nucleic acid detection technology.CRISPR-Cas12 has signal am...The COVID-19 pandemic has brought great challenges to traditional nucleic acid detection technology.Thus,it is urgent to develop a more simple and efficient nucleic acid detection technology.CRISPR-Cas12 has signal amplification ability,high sensitivity and high nucleic acid recognition specificity,so it is considered as a nucleic acid detection tool with broad development prospects and high application value.This review paper discusses recent advances in CRISPR-Cas12-based nucleic acid detection,with an emphasis on the new research methods and means to improve the nucleic acid detection capability of CRISPR-Cas12.Strategies for improving sensitivity,optimization of integrated detection,development of sim-plified detection mode and improvement of quantitative detection capabilities are included.Finally,the future development of CRISPR-Cas12-based nucleic acids detection is prospected.展开更多
A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently d...A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently developed for detecting nucleic acids, however, are often with expensive, complicated and timeconsuming preparation process. Here, by using a facile bottom-up synthesis method, a two-dimensional(2 D) coordination polymer(CP) nanosheet, [Cu(tz)](Htz = 1,2,4-triazole), was successfully prepared after optimizing reaction conditions. These ultrathin CP nanosheets with thickness of 4.7 ± 1.1 nm could readily form nanosensors by assembly with DNA probes, which exhibited a low limit of detection(LOD)for p53 DNA fragment as 144 pmol/L. Furthermore, by integrating [Cu(tz)] nanosheets with hybridization chain reaction(HCR) probes, mi R-21, one kind of micro RNA upregulated in many cancer cells, can be sensitively detected with a LOD of 100 pmol/L and monitored in living cells, giving consistent results with those obtained by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) analysis.Thus [Cu(tz)] nanosheets, which not only possess much better nucleic acids sensing performance than bulk cystals, but also exhibit nucleic acid delivery functions, could be used as a novel nanoplatform in biomedical imaging and sensing applications.展开更多
Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real...Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),the gold-standard method,still has shortfalls in diagnostic sensitivity and timeliness.Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay(RT-RAP)to detect HEV fragment within an hour.The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains.Among 15 types of HEV(species A-C),the sensitivity of RT-RAP was approximately 2-8 folds lower than that of the qRT-PCR in 9 types,and no-cross reaction with other viruses was observed.RT-RAP was further applied to analyze CSF and fecal specimens;the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results.These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.展开更多
The outbreak of COVID-19 has drawn great attention around the world.SARS-CoV-2 is a highly infectious virus with occult transmission by many mutations and a long incubation period.In particular,the emergence of asympt...The outbreak of COVID-19 has drawn great attention around the world.SARS-CoV-2 is a highly infectious virus with occult transmission by many mutations and a long incubation period.In particular,the emergence of asymptomatic infections has made the epidemic even more severe.Therefore,early diagnosis and timely management of suspected cases are essential measures to control the spread of the virus.Developing simple,portable,and accurate diagnostic techniques for SARS-CoV-2 is the key to epidemic prevention.The advantages of point-of-care testing technology make it play an increasingly important role in viral detection and screening.This review summarizes the point-of-care testing platforms developed by nucleic acid detection,immunological detection,and nanomaterial-based biosensors detection.Furthermore,this paper provides a prospect for designing future highly accurate,cheap,and convenient SARS-CoV-2 diagnostic technology.展开更多
For some Cas nucleases,trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection.Portable single and multiplex nucleic acid-b...For some Cas nucleases,trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection.Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture.Here,we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool.We systematically characterized AsCas12a,LbCas12a,LwaCas13a,and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection.Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input.Using this tool,we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field.Our method,from sample preparation to result readout,could be rapidly and easily deployed in the field.This system could be extended to other crop pathogens,including those that currently lack a detection method and have metabolite profiles that make detection challenging.This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping,transgene detection,and qualitative detection of gene expression in the field.展开更多
Background:A patient’s infectivity is determined by the presence of the virus in different body fluids,secretions,and excreta.The persistence and clearance of viral RNA from different specimens of patients with 2019 ...Background:A patient’s infectivity is determined by the presence of the virus in different body fluids,secretions,and excreta.The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease(COVID-19)remain unclear.This study analyzed the clearance time and factors influencing 2019 novel coronavirus(2019-nCoV)RNA in different samples from patients with COVID-19,providing further evidence to improve the management of patients during convalescence.Methods:The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20,2020 to February 10,2020 were collected retrospectively.The reverse transcription polymerase chain reaction(RT-PCR)results for patients’oropharyngeal swab,stool,urine,and serum samples were collected and analyzed.Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive(minimum 24 h sampling interval)negative RT-PCR results for viral RNA from oropharyngeal swabs.The effects of cluster of differentiation 4(CD4)+T lymphocytes,inflammatory indicators,and glucocorticoid treatment on viral nucleic acid clearance were analyzed.Results:In the 292 confirmed cases,66 patients recovered after treatment and were included in our study.In total,28(42.4%)women and 38 men(57.6%)with a median age of 44.0(34.0-62.0)years were analyzed.After in-hospital treatment,patients’inflammatory indicators decreased with improved clinical condition.The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5(6.0-11.0)days.By February 10,2020,11 convalescent patients(16.7%)still tested positive for viral RNA from stool specimens and the other 55 patients’stool specimens were negative for 2019-nCoV following a median duration of 11.0(9.0-16.0)days after symptom onset.Among these 55 patients,43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs,with a median delay of 2.0(1.0-4.0)days.Results for only four(6.9%)urine samples were positive for viral nucleic acid out of 58 cases;viral RNA was still present in three patients’urine specimens after throat swabs were negative.Using a multiple linear regression model(F=2.669,P=0.044,and adjusted R2=0.122),the analysis showed that the CD4+T lymphocyte count may help predict the duration of viral RNA detection in patients’stools(t=-2.699,P=0.010).The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group(15 days vs.8.0 days,respectively;t=2.550,P=0.013)and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group(20 days vs.11 days,respectively;t=4.631,P<0.001).There was no statistically significant difference in inflammatory indicators between patients with positive fecal viral RNA test results and those with negative results(P>0.05).Conclusions:In brief,as the clearance of viral RNA in patients’stools was delayed compared to that in oropharyngeal swabs,it is important to identify viral RNA in feces during convalescence.Because of the delayed clearance of viral RNA in the glucocorticoid treatment group,glucocorticoids are not recommended in the treatment of COVID-19,especially for mild disease.The duration of RNA detection may relate to host cell immunity.展开更多
文摘Objective: The objective of the study is to verify the clinical validity of the following kits with the comparative experimental analysis and evaluate whether their performance can meet the clinical requirements, i.e. Class III in vitro diagnostic reagent “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (PCR-Fluorescence Probe Method)” of Daan Gene Co., Ltd. (Daan kit for short) and “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (Fluorescence PCR Method)” of Wuhan Biot Gene Co., Ltd. (Biot kit for short). Method: In the study process, the samples were divided into positive and negative groups according to the control test results, and the clinical application performance of Daan kit and Biot kit was evaluated by comparing their test results. Results: The results show that two kits indicate the same test results, i.e. 26 positive and 107 negative samples in a total of 133 male urethral discharge samples, and 32 positive and 238 negative samples in a total of 270 female cervical secretion samples. Conclusion: It can be concluded from the clinical test that Daan and Biot Herpes Simplex Virus (HSV) Type II Nuc- leic Acid Test Kits are reliable, accurate, safe, convenient for use, stable and high-value in the clinical application.
基金the Science and Technology Joint Project of the Yangtze River Delta (19395810100)Shanghai Agriculture Project (19391901500)+2 种基金the National Key Research and Development Program of China (2016YFD0501101)the Shanghai Science and Technology Innovation Foundation (19441903900 and 19XD1433000)Project of National Natural Science Foundation of China (82003705)。
文摘The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.
基金supported by the National Natural Science Foundation of China(31900060)National Key Research and Development Program of China(2020YFA0907800,2022YFC3400200,2022YFA0912200)+1 种基金Natural Science Foundation of Shanghai(20ZR1414500)Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University(21TQ1400204).
文摘Nucleic acid detection plays a key role in diverse diagnosis and disease control.Currently available nucleic acid detection techniques are challenged by trade-offs among speed,simplicity,precision and cost.Here,we described a novel method,designated SENSOR(Sulfur DNA mediated nucleic acid sensing platform),for rapid nucleic acid detection.SENSOR was developed from phosphorothioate(PT)-DNA and sulfur binding domain(SBD)which specifically binds double-stranded PT-modified DNA.SENSOR utilizes PT-DNA oligo and SBD as targeting module,which is linked with split luciferase reporter to generate luminescence signal within 10 min.We tested detection on synthesized nucleic acid and COVID-19 pseudovirus,achieving attomolar sensitivity combined with an amplification procedure.Single nucleotide polymorphisms(SNP)could also be discriminated.Indicating SENSOR a new promising nucleic acid detection technique.
基金supported by the National Major Scientific and Technological Special Project(no.2012ZX10004220)the National Natural Science Foundation of China(nos.81401688,81271855,81261160324,81371836,and 81572023)。
文摘Schistosomiasis is an important zoonotic parasitic disease that causes serious harms to humans and animals.Surveillance and diagnosis play key roles in schistosomiasis control,however,current techniques for surveillance and diagnosis of the disease have limitations.As genome data for parasites are increasing,novel techniques for detection incorporating nucleotide sequences are receiving widespread attention.These sensitive,specific,and rapid detection methods are particularly important in the diagnosis of low-grade and early infections,and may prove to have clinical significance.This paper reviews the progress of nucleic acid detection in the diagnosis and prevention of schistosomiasis,including such aspects as the selection of target genes,and development and application of nucleic acid detection methods.
基金supported by the Science and Technology Program of Fujian Province (2018Y4013 to B.-A.L.)the Science and Technology Project of Xiamen Science and Technology Bureau (3502Z20193023 to B.-A.L.)+4 种基金the Health-Education Joint Research Project of Fujian Province (2019-WJ-34 to B.-A.L. and Z.-M.Z)the COVID-19 Emergency Research Project of Xiamen Science and Technology Bureau (3502Z2020YJ21 to Bio Detect (Xiamen) Biotechnology Co., Ltd.)the COVID-19 Emergency Research Project of Xiamen University (X2106103 to B.-A.L.)the National Natural Science Foundation of China (U1705284, 81972458, and 81772958 to B.-A.L.)Project 111 sponsored by the State Bureau of Foreign Experts and Ministry of Education (B06016)。
文摘Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to prevent the further spread of the epidemic. An optimized RPA-Cas12a-based platform combined with digital microfluidics(DMF), the RCD platform, was established to achieve the automated, rapid detection of influenza viruses and SARS-CoV-2. The probe in the RPA-Cas12a system was optimized to produce maximal fluorescence to increase the amplification signal. The reaction droplets in the platform were all at the microliter level and the detection could be accomplished within 30 min due to the effective mixing of droplets by digital microfluidic technology. The whole process from amplification to recognition is completed in the chip, which reduces the risk of aerosol contamination. One chip can contain multiple detection reaction areas, offering the potential for customized detection.The RCD platform demonstrated a high level of sensitivity, specificity(no false positives or negatives), speed(≤30 min),automation and multiplexing. We also used the RCD platform to detect nucleic acids from influenza patients and COVID-19 patients. The results were consistent with the findings of q PCR. The RCD platform is a one-step, rapid, highly sensitive and specific method with the advantages of digital microfluidic technology, which circumvents the shortcomings of manual operation. The development of the RCD platform provides potential for the isothermal automatic detection of nucleic acids during epidemics.
基金Natural Science Foundation of Hubei Province,China,No.2016CFB596and Wuhan City Medical Research Project,China,No.WX17Q39 and No.WX15B14.
文摘BACKGROUND The global outbreak of human severe acute respiratory syndrome coronavirus(SARS-CoV)-2 infection represents an urgent need for readily available,accurate and rapid diagnostic tests.Nucleic acid testing of respiratory tract specimens for SARS-CoV-2 is the current gold standard for diagnosis of coronavirus disease 2019(COVID-19).However,the diagnostic accuracy of reverse transcription polymerase chain reaction(RT-PCR)tests for detecting SARS-CoV-2 nucleic acid may be lower than optimal.The detection of SARS-CoV-2-specific antibodies should be used as a serological non-invasive tool for the diagnosis and management of SARS-CoV-2 infection.AIM To investigate the diagnostic value of SARS-CoV-2 IgM/IgG and nucleic acid detection in COVID-19.METHODS We retrospectively analyzed 652 suspected COVID-19 patients,and 206 non-COVID-19 patients in Wuhan Integrated TCM and Western Medicine Hospital.Data on SARS-CoV-2 nucleic acid tests and serum antibody tests were collected to investigate the diagnostic value of nucleic acid RT-PCR test kits and immunoglobulin(Ig)M/IgG antibody test kits.The j2 test was used to compare differences between categorical variables.A 95%confidence interval(CI)was provided by the Wilson score method.All analyses were performed with IBM SPSS Statistics version 22.0(IBM Corp.,Armonk,NY,United States).RESULTS Of the 652 suspected COVID-19 patients,237(36.3%)had positive nucleic acid tests,311(47.7%)were positive for IgM,and 592(90.8%)were positive for IgG.There was a significant difference in the positive detection rate between the IgM and IgG test groups(P<0.001).Using the RT-PCR results as a reference,the specificity,sensitivity,and accuracy of IgM/IgG combined tests for SARS-CoV-2 infection were 98.5%,95.8%,and 97.1%,respectively.Of the 415 suspected COVID-19 patients with negative nucleic acid test results,366 had positive IgM/IgG tests with a positive detection rate of 88.2%.CONCLUSION Our data indicate that serological IgM/IgG antibody combined test had high sensitivity and specificity for the diagnosis of SARS-CoV-2 infection,and can be used in combination with RT-PCR for the diagnosis of SARS-CoV-2 infection.
文摘Nucleic acid detection,widely used in clinical diagnosis,biological analysis,and environmental monitoring,is of great significance for disease diagnosis and basic research.With the outbreak of COVID-19,the demand for fast and high-throughput nucleic acid detection from large numbers of samples has increased sharply.Automated nucleic acid detection systems can meet these needs,and also play important roles in disease screening and infectious disease prevention and control.In this review,we introduce and compare the current mainstream nucleic acid automatic detection instruments and equipment,then discuss the future demands of nucleic acid detection.
基金This research was financially supported by the National Natural Science Foundation of China(NSFC,No.62071119)the Open Funding of State Key Laboratory of Oral Diseases(No.SKLOD2022OF05)+1 种基金the Jiangsu Provincial Key Research and Development Program(No.BA2020016)the National Fund(No.BWS19C016).
文摘Nucleic acid detection(NAD)based on real-time polymerase chain reaction(real-time PCR)is gold standard for infectious disease detection.Magnetic nanoparticles(MNPs)are widely used for nucleic acid extraction(NAE)because of their excellent properties.Microfluidic technology makes automated NAD possible.However,most of the NAD microfluidic chips are too complex to be applied to point-of-care(POC)testing.In this paper,a simple-structure cartridge was developed for POC detection of infectious diseases.This self-contained cartridge can be divided into a magnetic-controlled NAE part,a valve-piston combined fluidic control part and a PCR chip,which is able to extract nucleic acid from up to 500μL of liquid samples by MNPs and finish the detection process from“sample in”to“answer out”automatically.Performance tests of the cartridges show that it met the demands of automated NAD.Results of on-cartridge detection of hepatitis B virus(HBV)demonstrated that this system has good uniformity and no cross-contamination between different cartridges,and the limit of detection(LOD)of this system for HBV in serum is 50 IU/mL.Multiplex detections of severe acute respiratory syndrome coronaviruses 2(SARS-CoV-2)with a concentration of 500 copies/mL were carried out on the system and 100%positive detection rate was achieved.
基金supported by the National Natural Science Foundation of China(project No.81970029)Fundamental Research Funds for the Central Universities of China(The Emergency Projects on COVID-19,xzy032020042)Qinnong Bank-XJTU special project for COVID-19(qnxjtu-12)。
文摘Coronavirus disease 2019(COVID-19)has been a pandemic for more than a year.With the expanding second wave of the pandemic in winter,the continuous evolution of SARS-CoV-2 has brought new issues,including the significance of virus mutations in infection and the detection of asymptomatic infection.In this review,we first introduced several major SARS-CoV-2 mutations since the COVID-19 outbreak and then mentioned the widely used molecular detection techniques to diagnose COVID-19,primarily focusing on their strengths and limitations.We further discussed the effects of viral genetic variation and asymptomatic infection on the molecular detection of SARS-CoV-2 infection.The review finally summarized useful insights into the molecular diagnosis of COVID-19 under the special situation being challenged by virus mutation and asymptomatic infection.
文摘Objective: The study aims to understand the characteristics and epidemic trend of the pathogen of hand, foot and mouth disease (HFMD) in Guangxi regions, China. Besides, it aims to analyze the differences of intestinal virus detection rate between anal swab and pharyngeal swab samples. Methods: Anal swab and pharyngeal swabs of suspected HFMD children were collected in our hospital from 2012 to 2015. Real-time fluorescent PCR (Polymerase Chain Reaction) was used to detect enterovirus 71 (EV71), coxsackie virus type 16 (CA16), and universal intestinal virus nucleic acid (EV). Composition and conversion of predominant pathogens were analyzed, and paired samples’ test results of swabs anal and pharyngeal swab were statistically analyzed. Results: There are 681 cases with enterovirus in 2351 cases of patients. Among those who got enterovirus, there are 501 cases of EV71, 102 cases of CA16 and 79 cases of EV. From 2012 to 2015, the total proportion of the virus detection is 46.47%, 16.23%, 41.02% and 15.33% respectively in each year, while the proportion of predominant epidemic virus is 93.93% of EV71, 66.12% of CA16, 89.30% of EV71 and 98.73% of EV, non-EV71, non-CA16 EV (from October to December in 2015). It’s obvious that the total virus detection rate in 2012 and 2014 is significantly higher than that in 2013 and 2015. There is statistical significance. Conclusion: The main HFMD pathogens are EV71 from 2012 to 2015 in Guangxi regions. In 2012 and 2014, the predominant epidemic pathogens were EV71, while in 2013 and 2015, the predominant epidemic pathogens turn to be CA16 and non-EV71, non-CA16 EV respectively. What’s more, collecting anal swab and pharyngeal swab virus at the same time for nucleic acid detection is of great significance to improve the HFMD laboratory diagnostic.
基金supported by the National Natural Science Foundation of China(91959128,21874049).
文摘The COVID-19 pandemic has brought great challenges to traditional nucleic acid detection technology.Thus,it is urgent to develop a more simple and efficient nucleic acid detection technology.CRISPR-Cas12 has signal amplification ability,high sensitivity and high nucleic acid recognition specificity,so it is considered as a nucleic acid detection tool with broad development prospects and high application value.This review paper discusses recent advances in CRISPR-Cas12-based nucleic acid detection,with an emphasis on the new research methods and means to improve the nucleic acid detection capability of CRISPR-Cas12.Strategies for improving sensitivity,optimization of integrated detection,development of sim-plified detection mode and improvement of quantitative detection capabilities are included.Finally,the future development of CRISPR-Cas12-based nucleic acids detection is prospected.
基金supported by the National Key Research and Development Program of China (No.2018YFA0902801)the National Natural Science Foundations of China (Nos.21775169,21801259 and 21974153)+4 种基金the Scientific Technology Project of Shenzhen City (No.JCYJ20200109142410170)the Scientific Technology Project of Guangzhou City (No.202103000003)the Guangdong Natural Science Foundation (Nos.2018A030313290,2019A1515010587)the Guangdong Science and Technology Plan Project (No.2020B1212060077)the Fundamental Research Funds for the Central Universities,SYSU (No.19lgpy142)。
文摘A reliable and sensitive strategy which can assess nucleic acid levels in living cells would be essential for fundamental research of biomedical applications. Some nanomaterial-based fluorescence biosensors recently developed for detecting nucleic acids, however, are often with expensive, complicated and timeconsuming preparation process. Here, by using a facile bottom-up synthesis method, a two-dimensional(2 D) coordination polymer(CP) nanosheet, [Cu(tz)](Htz = 1,2,4-triazole), was successfully prepared after optimizing reaction conditions. These ultrathin CP nanosheets with thickness of 4.7 ± 1.1 nm could readily form nanosensors by assembly with DNA probes, which exhibited a low limit of detection(LOD)for p53 DNA fragment as 144 pmol/L. Furthermore, by integrating [Cu(tz)] nanosheets with hybridization chain reaction(HCR) probes, mi R-21, one kind of micro RNA upregulated in many cancer cells, can be sensitively detected with a LOD of 100 pmol/L and monitored in living cells, giving consistent results with those obtained by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) analysis.Thus [Cu(tz)] nanosheets, which not only possess much better nucleic acids sensing performance than bulk cystals, but also exhibit nucleic acid delivery functions, could be used as a novel nanoplatform in biomedical imaging and sensing applications.
基金This work was supported by the National Key R&D Program of China(2021YFC2301102)National Natural Science Foundation of China(82202593)the Central Guidance on Local Science and Technology Development Fund of Hebei Province(216Z7713G).
文摘Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),the gold-standard method,still has shortfalls in diagnostic sensitivity and timeliness.Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay(RT-RAP)to detect HEV fragment within an hour.The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains.Among 15 types of HEV(species A-C),the sensitivity of RT-RAP was approximately 2-8 folds lower than that of the qRT-PCR in 9 types,and no-cross reaction with other viruses was observed.RT-RAP was further applied to analyze CSF and fecal specimens;the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results.These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.
基金supported by the National Key R&D Program of China(No.2021YFC2301100)the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDB30000000)+3 种基金the National Natural Science Foundation of China(No.61890940)the Chongqing Bayu Scholar Program(No.DP2020036)Program of Shanghai Academic Research Leaders(No.23XD1420200)Fudan University。
文摘The outbreak of COVID-19 has drawn great attention around the world.SARS-CoV-2 is a highly infectious virus with occult transmission by many mutations and a long incubation period.In particular,the emergence of asymptomatic infections has made the epidemic even more severe.Therefore,early diagnosis and timely management of suspected cases are essential measures to control the spread of the virus.Developing simple,portable,and accurate diagnostic techniques for SARS-CoV-2 is the key to epidemic prevention.The advantages of point-of-care testing technology make it play an increasingly important role in viral detection and screening.This review summarizes the point-of-care testing platforms developed by nucleic acid detection,immunological detection,and nanomaterial-based biosensors detection.Furthermore,this paper provides a prospect for designing future highly accurate,cheap,and convenient SARS-CoV-2 diagnostic technology.
基金supported by the National Natural Science Foundation of China(31771808 and 32001551)the China Postdoctoral Science Foundation(2020M680779)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(S2021ZD03)。
文摘For some Cas nucleases,trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection.Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture.Here,we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool.We systematically characterized AsCas12a,LbCas12a,LwaCas13a,and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection.Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input.Using this tool,we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field.Our method,from sample preparation to result readout,could be rapidly and easily deployed in the field.This system could be extended to other crop pathogens,including those that currently lack a detection method and have metabolite profiles that make detection challenging.This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping,transgene detection,and qualitative detection of gene expression in the field.
基金The work was supported by grants from the First-class university and first-class discipline building project of the Fudan University(No.IDF162005)the Scientific research for special subjects on 2019 novel coronavirus(No.2019-nCoV)the Shanghai Public Health Clinical Center(No.2020YJKY01)。
文摘Background:A patient’s infectivity is determined by the presence of the virus in different body fluids,secretions,and excreta.The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease(COVID-19)remain unclear.This study analyzed the clearance time and factors influencing 2019 novel coronavirus(2019-nCoV)RNA in different samples from patients with COVID-19,providing further evidence to improve the management of patients during convalescence.Methods:The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20,2020 to February 10,2020 were collected retrospectively.The reverse transcription polymerase chain reaction(RT-PCR)results for patients’oropharyngeal swab,stool,urine,and serum samples were collected and analyzed.Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive(minimum 24 h sampling interval)negative RT-PCR results for viral RNA from oropharyngeal swabs.The effects of cluster of differentiation 4(CD4)+T lymphocytes,inflammatory indicators,and glucocorticoid treatment on viral nucleic acid clearance were analyzed.Results:In the 292 confirmed cases,66 patients recovered after treatment and were included in our study.In total,28(42.4%)women and 38 men(57.6%)with a median age of 44.0(34.0-62.0)years were analyzed.After in-hospital treatment,patients’inflammatory indicators decreased with improved clinical condition.The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5(6.0-11.0)days.By February 10,2020,11 convalescent patients(16.7%)still tested positive for viral RNA from stool specimens and the other 55 patients’stool specimens were negative for 2019-nCoV following a median duration of 11.0(9.0-16.0)days after symptom onset.Among these 55 patients,43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs,with a median delay of 2.0(1.0-4.0)days.Results for only four(6.9%)urine samples were positive for viral nucleic acid out of 58 cases;viral RNA was still present in three patients’urine specimens after throat swabs were negative.Using a multiple linear regression model(F=2.669,P=0.044,and adjusted R2=0.122),the analysis showed that the CD4+T lymphocyte count may help predict the duration of viral RNA detection in patients’stools(t=-2.699,P=0.010).The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group(15 days vs.8.0 days,respectively;t=2.550,P=0.013)and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group(20 days vs.11 days,respectively;t=4.631,P<0.001).There was no statistically significant difference in inflammatory indicators between patients with positive fecal viral RNA test results and those with negative results(P>0.05).Conclusions:In brief,as the clearance of viral RNA in patients’stools was delayed compared to that in oropharyngeal swabs,it is important to identify viral RNA in feces during convalescence.Because of the delayed clearance of viral RNA in the glucocorticoid treatment group,glucocorticoids are not recommended in the treatment of COVID-19,especially for mild disease.The duration of RNA detection may relate to host cell immunity.
