Study on the correlation between abdominal infection and psychological stress in children based on nucleic acid detection

BACKGROUND Diagnosing and treating abdominal infection in children remains a challenge.Nucleic acid detection,as a rapid and accurate diagnosis tool,has great significance in this field.AIM To investigate the diagnosis and treatment of abdominal infection by nucleic acid detection and its possible correlation with psychological stress in children.METHODS A total of 50 pediatric patients diagnosed with abdominal infections between September 2020 and July 2021 were included in this study.Intra-abdominal pus samples were collected for pathogen culture,drug susceptibility testing,and broad-spectrum bacterial nucleic acid testing.Psychological stress,anxiety,depression,and coping styles were assessed using the coping with a disease(CODI)scale.RESULTS Based on susceptibility testing,a regimen of cefazoxime,piperacillin/tazobactam,and metronidazole or ornidazole achieved 100%effectiveness in treating appendicitis.Psychological assessments revealed a positive correlation between pressure level and both anxiety(r=0.324,P=0.001)and depressive disorders(r=0.325,P<0.001).Acceptance and distancing as coping strategies were negatively correlated with anxiety and depression,while negative emotional responses were strongly associated with increased anxiety(r=0.574,P<0.001)and depression(r=0.511,P=0.001).Coping strategies such as illusion and escape showed no significant correlation with emotional outcomes.CONCLUSION Nucleic acid testing helps in the diagnosis of abdominal infections in children,and also focuses on children's mental health.

Consistency Analysis of Detection Results of Two Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kits

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.

Development of an Integrated Disposable Device for SARSCoV-2 Nucleic Acid Extraction and Detection

Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods We designed,developed,and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection.The precision of the liquid transfer and temperature control was tested.A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction(RT-PCR).The entire process,from SARS-CoV-2 nucleic acid extraction to amplification,was evaluated.Results The precision of the syringe transfer volume was 19.2±1.9μL(set value was 20),32.2±1.6(set value was 30),and 57.2±3.5(set value was 60).Temperature control in the amplification tube was measured at 60.0±0.0℃(set value was 60)and 95.1±0.2℃(set value was 95)respectively.SARS-Cov-2 nucleic acid extraction yield through the device was 7.10×10^(6) copies/mL,while a commercial kit yielded 2.98×10^(6) copies/mL.The mean time to complete the entire assay,from SARS-CoV-2 nucleic acid extraction to amplification detection,was 36 min and 45 s.The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL.Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test(POCT).

Nucleic acid detection in the diagnosis and prevention of schistosomiasis

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.

An automated nucleic acid detection platform using digital microfluidics with an optimized Cas12a system

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.

Ultrathin metal-organic framework nanosheets (Cu-TCPP)-based isothermal nucleic acid amplification for food allergen detection

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.

Recent developments and trends of automatic nucleic acid detection systems

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.

Clinical application of combined detection of SARS-CoV-2-specific antibody and nucleic acid

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.

A rapid nucleic acid detection platform based on phosphorothioate-DNA and sulfur binding domain

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.

Molecular detection of SARS-CoV-2 being challenged by virus variation and asymptomatic infection

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.

Results on Pathogen Detection of Foot and Mouth Disease in Guangxi China and Analysis on Its Popular Spectrum

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.

Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

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.

Improving treatment plan and mental health in children with abdominal infection for broad-spectrum bacterial infections

BACKGROUND Pediatric abdominal infection is a common but serious disease that requires timely and effective treatment.In surgical treatment,accurate diagnosis and rational application of antibiotics are the keys to improving treatment effects.AIM To investigate the effect of broad-spectrum bacterial detection on postoperative antibiotic therapy.METHODS A total of 100 children with abdominal infection who received surgical treatment in our hospital from September 2020 to July 2021 were grouped.The observation group collected blood samples upon admission and sent them for broad-spectrum bacterial infection nucleic acid testing,and collected pus or exudate during the operation for bacterial culture and drug sensitivity testing;the control group only sent bacterial culture and drug sensitivity testing during the operation.RESULTS White blood cell count,C-reactive protein,procalcitonin,3 days after surgery,showed better postoperative index than the control group(P<0.05).The hospital stay in the observation group was significantly shorter than that in the control group.The hospitalization cost in the observation group was significantly lower than that in the control group,and the difference between the two groups was statistically significant(P<0.05).CONCLUSION Early detection of broad-spectrum bacterial infection nucleic acids in pediatric abdominal infections can help identify pathogens sooner and guide the appropriate use of antibiotics,improving treatment outcomes and reducing medical costs to some extent.

A novel cartridge for nucleic acid extraction,amplification and detection of infectious disease pathogens with the help of magnetic nanoparticles

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.

Two-dimensional coordination polymer-based nanosensor for sensitive and reliable nucleic acids detection in living cells

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.

超高并发核酸检测管理系统的设计与实现

着重介绍核酸检测管理系统在低成本下处理高并发业务的建设难点、功能实现、应用成效,旨在为相同应用场景提供借鉴及参考。基于千万级人口核酸检测中的极端超高并发,针对采样现场网络易拥堵、“平急”并发量差异悬殊、转运过程难追踪等实际问题,采取“前置计算、静态提供、用后更新”的核酸码生成模式,解决突发应急状态下的超高并发压力。通过“登记码”“样本编码”“转运箱码”“封箱码”的关联与管理,规范登记、采样、转运、接收、检测、查询各业务要素的责任主体与职责边界,建立高效运作的业务闭环。

江苏省新型冠状病毒核酸检测实验室能力建设调查

目的了解江苏省各级疾病预防控制机构应对新型冠状病毒的实验室能力情况,为优化省、市和区(县)生物安全实验室网络布局、满足新型冠状病毒肺炎等重大疫情防控多层次检测需求提供政策依据。方法根据新型冠状病毒检测所需的实验室仪器、专业技术人员、实验室备案与质量控制等关键指标制定调查表进行调查,并对调查结果进行统计学分析。结果江苏省共有疾病预防控制机构112家,配备生物安全实验室124个,核酸提取、定量聚合酶链式反应(polymerase chain reaction,PCR)仪器分别为85和112台,核酸检测专业人员有249人,仅有29个实验室完成PCR备案,全省每日最大检测样本数为5236个,占理论值的74.0%。结论江苏省新冠病毒核酸检测相关的实验室能力尚不均衡。省、市两级生物安全实验室能力建设较好,区(县)级较为薄弱,区(县)级疾病预防控制机构普遍存在仪器设备配备不足和检测人员缺乏等问题,具备核酸检测能力的机构较少;区域资源配置不均衡,苏北地区硬件设施和人员配置相对不足,具备核酸检测能力的机构较少;全省疾病预防控制机构的实验室均存在PCR备案和质量控制管理不完善的问题。

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

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.

A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

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.