Tb^(3+)-nucleic acid probe-based label-free and rapid detection of mercury pollution in food

Mercury is a threatening pollutant in food,herein,we developed a Tb^(3+)-nucleic acid probe-based label-free assay for mix-and-read,rapid detection of mercury pollution.The assay utilized the feature of light-up fluorescence of terbium ions(Tb^(3+))via binding with single-strand DNA.Mercury ion,Hg^(2+)induced thymine(T)-rich DNA strand to form a double-strand structure(T-Hg^(2+)-T),thus leading to fluorescence reduction.Based on the principle,Hg^(2+)can be quantified based on the fluorescence of Tb^(3+),the limit of detection was 0.0689μmol/L and the linear range was 0.1-6.0μmol/L.Due to the specificity of T-Hg^(2+)-T artificial base pair,the assay could distinguish Hg^(2+)from other metal ions.The recovery rate was ranged in 98.71%-101.34%for detecting mercury pollution in three food samples.The assay is low-cost,separation-free and mix-to-read,thus was a competitive tool for detection of mercury pollution to ensure food safety.

Recent advances in living cell nucleic acid probes based on nanomaterials for early cancer diagnosis

The early diagnosis of cancer is vital for effective treatment and improved prognosis. Tumor biomarkers, which can be used for the early diagnosis, treatment, and prognostic evaluation of cancer, have emerged as a topic of intense research interest in recent years. Nucleic acid, as a type of tumor biomarker, contains vital genetic information, which is of great significance for the occurrence and development of cancer. Currently, living cell nucleic acid probes, which enable the in situ imaging and dynamic monitoring of nucleic acids, have become a rapidly developing field. This review focuses on living cell nucleic acid probes that can be used for the early diagnosis of tumors. We describe the fundamental design of the probe in terms of three units and focus on the roles of different nanomaterials in probe delivery.

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).

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.

Spherical nucleic acids for biomedical applications

Spherical nucleic acids(SNAs)are a 3D spherical nanostructure composed of highly oriented,dense layers of oligonucleotides conjugated to a hollow or solid core.This structure allows SNAs to show resistance to nuclease degradation,enter into nearly all cells without transfection agents and enable precise interactions with target molecules.Based on superior biological properties,SNAs can be tailored for diverse biological applications,rendering them a flexible and biosafe tool for biological applications as well as an enabling platform for therapy.In this review,we mainly discuss the structure and conjugation mode of SNAs and focus on recent advances in their applications,such as biomedical detection,imaging,and drug delivery.Finally,the remaining challenges and future directions of SNAs are also discussed and proposed.

Comparison of Nucleic Acid Content among Non-diapause Pupae, Diapause Pupae and Eclosion-adult from Diapause Pupae of Papilio memnon

[Objective] The aim of this study was to provide basis for deeply understanding the diapause mechanism of Papilio memnon L. [Method] RNA and DNA content of non-diapause pupae, diapause pupae and eclosion-adult from diapause pupae at different development stages were detected by the colorimetry. [Result] RNA content of non-diapause pupae was 4.614 0-7.946 3 μg/mg, while diapause pupae was 4.326 0-5.885 3 μg/mg and eclosion-adult from diapause pupae was 20.779 3 μg/mg at initial stage. DNA content of non-diapause pupae was 0.448 7-0.535 0 μg/mg, while diapause pupae was 0.452 0-0.828 3 μg/mg and eclosion-adult from diapause pupae was 1.727 0 μg/mg at initial stage. [Conclusion] The nucleic acid content and change is related to the development stage.

Real-Time PCR Technique and Its Application in Quantification of Plant Nucleic Acid Molecules

Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.

Recent advances and perspectives of nucleic acid detection for coronavirus

The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments,saving people's lives and preventing epidemics.It is important to establish a quick standard diagnostic test for the detection of the infectious disease(COVID-19)to prevent subsequent secondary spread.Polymerase chain reaction(PCR)is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity.Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion.A variety of improved or new approaches also have been developed.This review summarizes the currently available detection methods for coronavirus nucleic acid.It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.

Liquid biopsy in patients with pancreatic cancer: Circulating tumor cells and cell-free nucleic acids

Despite recent advances in surgical techniques and perioperative management, the prognosis of pancreatic cancer(PCa) remains extremely poor. To provide optimal treatment for each patient with Pca, superior biomarkers are urgently needed in all phases of management from early detection to staging, treatment monitoring, and prognosis. In the blood of patients with cancer, circulating tumor cells(CTCs) and cell-free nucleic acids(cf NAs), such as DNA, m RNA, and noncoding RNA have been recognized. In the recent years, their presence in the blood has encouraged researchers to investigate their potential use as novel blood biomarkers, and numerous studies have demonstrated their potential clinical utility as a biomarker for certain types of cancer. This concept, called "liquid biopsy" has been focused on as a less invasive, alternative approach to cancer tissue biopsy for obtaining genetic and epigenetic aberrations that contribute to oncogenesis and cancer progression. In this article, we review the available literature on CTCs and cfN As in patients with cancer, particularly focusing on PCa, and discuss future perspectives in this field.

Design,delivery and efficacy testing of therapeutic nucleic acids used to inhibit hepatitis C virus gene expression in vitro and in vivo

Despite major achievements in the treatment ofchronic hepatitis C with the combination ofinterferons and the nucleoside analog ribavirin themajority of patients with chronic hepatitis C virus(HCV) infection cannot be treated effectively.Toimprove this response rate we used antisensetechnologies to inhibit HCV translation as possibleadditional option for experimental treatment.Antisense oligodeoxynucleotides(ODN) are

Mechanism of exogenous nucleic acids and their precursors improving the repair of intestinal epithelium after 7-irradiation in mice

AIM To clone expressed genes associated withrepair of irradiation-damaged mice intestinalgland cells treated by small intestinal RNA,andto explore the molecular mechanism ofexogenous nucleic acids improving repair ofintestinal crypt.METHODS The animal mode of test group andcontrol group was established,forty-five micebeing irradiated by γ ray were treated with smallintestinal RNA as test group,forty mice beingirradiated by γ ray were treated withphysiological saline as control group,five micewithout irradiation were used as normal control,their jejunal specimens were collectedrespectively at 6h,12h,24h,4d and 8d afterirradiation.Then by using LD-PCR based onsubtractive hybridization,these gene fragmentsdifferentially expressed between test group andcontrol group were obtained,and then werecloned into T vectors as well as beingsequenced.Obtained sequences were screenedagainst.GeneBank,if being new sequences,they were submitted to GeneBank.RESULTS Ninety clones were associated withrepair of irradiation-damaged intestinal glandcells treated by intestinal RNA.These clonesfrom test group of 6h,12h,24h,4d and 8dwere respectively 18,22,25,13,12.By screening against GeneBank,18 of which werenew sequences,the others were dramaticallysimilar to the known sequences,mainly similarto hsp,Nmi,Dutt1,alkaline phosphatase,homeobox,anti-CEA ScFv antibody,arginine/serine kinase and BMP-4,repA.Eighteen genefragments were new sequences,their acceptnumbers in GeneBank were respectivelyAF240164-AF240181.CONCLUSION Ninety clones were obtained tobe associated with repair of irradiation-damagedmice intestinal gland cells treated by smallintestinal RNA,which may be related toabnormal expression of genes and matchedproteins of hsp,Nmi,Duttl,Na,K-ATPase,alkalineph-osphatase,glkA,single strandedreplicative centromeric gene as well as 18 newsequences.

Liquid biopsy in patients with hepatocellular carcinoma: Circulating tumor cells and cell-free nucleic acids

Hepatocellular carcinoma(HCC), with its high incidence and mortality rate, is one of the most common malignant tumors. Despite recent development of a diagnostic and treatment method, the prognosis of HCC remains poor. Therefore, to provide optimal treatment for each patient with HCC, more precise and effective biomarkers are urgently needed which could facilitate a more detailed individualized decision-making during HCC treatment, including the following; risk assessment, early cancer detection, prediction of treatment or prognostic outcome. In the blood of cancer patients, accumulating evidence about circulating tumor cells and cell-free nucleic acids has suggested their potent clinical utilities as novel biomarker. This concept, so-called "liquid biopsy" is widely known as an alternative approach to cancer tissue biopsy. This method might facilitate a more sensitive diagnosis and better decision-making by obtaining genetic and epigenetic aberrations that are closely associated with cancer initiation and progression. In this article, we review recent developments based on the available literature on both circulating tumor cells and cell-free nucleic acids in cancer patients, especially focusing on Hepatocellular carcinoma.

Liquid biopsies for liquid tumors: emerging potential of circulating free nucleic acid evaluation for the management of hematologic malignancies

Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with hematologic malignancy cell free DNA reflects the underlying tumor mutational profile, whilst micro-RNAs reflect genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immune effector cells. These circulating nucleic acids offer a potentially simple, non-invasive, repeatable analysis that can aid in diagnosis, prognosis and therapeutic decisions in cancer treatment.

Liquid biopsy of gastric cancer patients:Circulating tumor cells and cell-free nucleic acids

To improve the clinical outcomes of cancer patients, early detection and accurate monitoring of diseases are necessary. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression, and analyses of these changes have been increasingly utilized for diagnostic, prognostic and therapeutic purposes in malignant diseases including gastric cancer (GC). Surgical and/or biopsy specimens are generally used to understand the tumor-associated alterations; however, those approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities, which may change during the therapeutic process. Therefore, the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized. This concept, so called &#x0201c;liquid biopsy&#x0201d;, would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of &#x0201c;tailor-made&#x0201d; cancer management programs. In the blood of cancer patients, the presence and potent utilities of circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) such as DNA, mRNA and microRNA have been recognized, and their clinical relevance is attracting considerable attention. In this review, we discuss recent developments in this research field as well as the relevance and future perspectives of CTCs and cfNAs in cancer patients, especially focusing on GC.

One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

Multi?targeted Antisense Oligonucleotide Delivery by a Framework Nucleic Acid for Inhibiting Biofilm Formation and Virulence

Biofilm formation is responsible for numerous chronic infections and represents a serious health challenge.Bacteria and the extracellular polysaccharides(EPS)cause biofilms to become adherent,toxic,resistant to antibiotics,and ultimately difficult to remove.Inhibition of EPS synthesis can prevent the formation of bacterial biofilms,reduce their robustness,and promote removal.Here,we have developed a framework nucleic acid delivery system with a tetrahedral configuration.It can easily access bacterial cells and functions by delivering antisense oligonucleotides that target specific genes.We designed antisense oligonucleotide sequences with multiple targets based on conserved regions of the VicK protein-binding site.Once delivered to bacterial cells,they significantly decreased EPS synthesis and biofilm thickness.Compared to existing approaches,this system is highly efficacious because it simultaneously reduces the expression of all targeted genes(gtfBCD,gbpB,ftf).We demonstrate a novel nucleic acid-based nanomaterial with multi-targeted inhibition that has great potential for the treatment of chronic infections caused by biofilms.

Antiviral effects of hepatitis B virus S gene-specific anti-gene locked nucleic acid in transgenic mice

AIM To assess the antiviral effects of hepatitis B virus(HBV) S gene-specific anti-gene locked nucleic acid(LNA) in transgenic mice.METHODS Thirty HBV transgenic mice were acclimatized to laboratory conditions and positive for serum HBV surface antigen(HBs Ag) and HBV DNA, were randomly divided into 5 groups(n = 7), including negative control(blank control, unrelated sequence control), positive control(lamivudine, anti-sense-LNA), and anti-gene-LNA experimental group. LNA was injected into transgenic mice by tail vein while lamivudine was administeredby gavage. Serum HBV DNA and HBs Ag levels were determined by fluorescence-based PCR and enzymelinked immune sorbent assay, respectively. HBV S gene expression amounts were assessed by reverse transcription polymerase chain reaction. Positive rates of HBsA g in liver cells were evaluated immunohistochemistry.RESULTS Average rate reductions of HBs Ag after treatment on the 3 rd, 5 th, and 7 th days were 32.34%, 45.96%, and 59.15%, respectively. The inhibitory effect of antigene-LNA on serum HBs Ag peaked on day 7, with statistically significant differences compared with pretreatment(0.96 ± 0.18 vs 2.35 ± 0.33, P < 0.05) and control values(P < 0.05 for all). Average reduction rates of HBV DNA on the 3 rd, 5 th, and 7 th days were 38.55%, 50.95%, and 62.26%, respectively. This inhibitory effect peaked on the 7 th day after treatment with anti-gene-LNA, with statistically significant differences compared with pre-treatment(4.17 ± 1.29 vs 11.05 ± 1.25, P < 0.05) and control values(P < 0.05 for all). The mR NA levels of the HBV S gene(P < 0.05 for all) and rates of HBsA g positive liver cells(P < 0.05 for all) were significantly reduced compared with the control groups. Liver and kidney function, and histology showed no abnormalities. CONCLUSION Anti-gene-LNA targeting the S gene of HBV displays strong inhibitory effects on HBV in transgenic mice, providing theoretical and experimental bases for gene therapy in HBV.

Application of cationic liposomes for delivery of nucleic acids

Nucleic acid-based bioactive substances have recently emerged as a new class of nextgeneration therapeutics, but their development has been limited by their relatively weakdelivery into target cells. Cationic liposomes have been studied as a means to enhance thestability of nucleic acid therapeutics in the bloodstream and improve their cellular delivery.As nucleic acid therapeutics, siRNA and plasmid DNA have been extensively tested fordelivery using cationic liposomes. This review discusses recent progress in the applicationof cationic liposomes for the delivery of nucleic acid therapeutics.

Construction and expression of nucleic acid vaccine pVAX1-h-alpha S_(1-140) coding human alpha-synuclein

BACKGROUND： The deposition of α -synuclein （ α -syn） aggregates is a neuropathological feature of Parkinson＇s disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson＇s disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson＇s disease. OBJECTIVE： To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells. DESIGN AND SETTING： The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University ＆ Chongqing Key Laboratory of Neurology. MATERIALS： The eukaryotic expression plasmid pVAXI, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA. METHODS： The full-length CDS sequence of the human a -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn Ⅰ, Xba Ⅰ and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn Ⅰ and Xba Ⅰ simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1-140 were transformed into competent E. coli TOP 1 0 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting. MAIN OUTCOME MEASURES： Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α-S1-140, and detection of the expression in mammalian cell COS-7. RESULTS： The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX 1-h α-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting. CONCLUSION： Nucleic acid vaccine pVAX1-h α S1-140 was successfully constructed and expressed in COS-7 cells.