Research progress in SARS-CoV-2 nucleic acid detection based on microfluidic platforms / 医疗卫生装备

The detection principle of microfluidic microfluidic technology was introduced.The current research status of microfluidic platform-based SARS-CoV-2 nucleic acid detection technologies were reviewed such as reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR),digital PCR,isothermal amplification and clustered regularly interspaced palindromic repeats/CRISPR-associated protein.The deficiencies of microfluidic platform-based SARS-CoV-2 nucleic acid detection were analyzed.It's pointed out microfluidic platform-based SARS-CoV-2 nucleic acid detection had to be optimized and validated clinically in specialty,sensitivity,detection limit,reproducibility,informatization,quality control and reagent cost.[Chinese Medical Equipment Journal,2024,45(1):101-107]

Application of the CRISPR/Cas system in gene editing and nucleic acid detection of parasitic diseases: a review / 中国血吸虫病防治杂志

CRISPR/Cas system, an adaptive immune system with clustered regularly interspaced short palindromic repeats, may interfere with exogenous nucleic acids and protect prokaryotes from external damages, is an effective gene editing and nucleic acid detection tools. The CRISPR/Cas system has been widely applied in virology and bacteriology; however, there is relatively less knowledge about the application of the CRISPR/Cas system in parasitic diseases. The review summarizes the mechanisms of action of the CRISPR/Cas system and provides a comprehensive overview of their application in gene editing and nucleic acid detection of parasitic diseases, so as to provide insights into future studies on parasitic diseases.

The Versatile Tool: CRISPR/Cas12a System for Nucleic Acid Detection / 生物化学与生物物理进展

The CRISPR/Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). The system forms an adaptive immune system in archaea and bacteria. The inherent defense mechanism enables these microorganisms to protect themselves against the invasion of foreign genetic material. The system functions of immune response including three main stages: adaptation, expression/maturation, and interference, each stage needs specific Cas proteins encoded by Cas gene located near the CRISPR sequences, along with other auxiliary proteins. In 2015, Zhang et al. reportedCas12a (Cpf1) as a member of the Class II type V CRISPR/Cas12a system, which possesses endonuclease activity. This finding holds great promise for its application in the field of biotechnology. In 2018, Doudna’s team first applied the CRISPR/Cas12a system for detecting HPV nucleic acid. The system comprises the following essential components in vitro detection: Cas12a, the crRNA sequence complementary to the target DNA, the PAM sequence, and the ssDNA reporter. Cas12a possesses a typical RuvC domain, displaying a canonical bilobed architecture that consists of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe contains the REC1 and REC2 domains, and the NUC lobe includes RuvC, PAM-interacting (PI), Wedge (WED), and bridge helix (BH) domains. The mature crRNA for Cas12a has a length of 42-44 nt, consists of repeat sequence (19/20 nt) and spacer sequence (23-25 nt). The crRNA spacer sequence has been found to require a length of 18 nt to achieve complete cleavage activity in vitro. Additionally, mutation in the bases of crRNA can indeed affect the activity of Cas12a. The PAM sequence plays a critical role in the recognition and degradation of DNA by the CRISPR/Cas system, enabling the system to distinguish between self and non-self genomic materials. Cas12a can effectively target the spacer sequence downstream of a T-rich PAM sequence at the 5' end. LbCas12a and AsCas12a both recognize the PAM sequences of 5'-TTTN-3', while FnCas12a recognizes the PAM sequences of 5'-TTN-3'. All of these PAM sequences are located upstream on the non-template strand (NTS) at the 5' end. Cas12a (Cpf1), guided by the crRNA, binds to the target DNA by recognizing the PAM sequence. It exhibits the ability to induce arbitrary cleavage of ssDNA within the system while cleaving the target ssDNA or dsDNA. According to this feature, an array of nucleic acid detection methods has been developed for tumor detection and infection diagnostics, such as the DETECTR (RPA-CRISPR/Cas12a method) and HOLMES (PCR-CRISPR/Cas12a method) in 2018. Then, in 2019, Cas12aVDet (one-step detection method), where Cas12a protein was immobilized on the upper wall of the reaction tube. This not only prevented contamination from opening the tube but also reduced the detection reaction time. In 2021, the dWS-CRISPR (digital warm-start CRISPR) was developed as a one-pot detection method. It serves as an accurate approach for quantitatively detectingSARS-CoV-2 in clinical specimens. With the innovation of scientific technology, the high-sensitivity signal transduction technology has also been integrated with the CRISPR/Cas12a system, enabling direct detection of nucleic acids, and eliminating the need for nucleic acid amplification steps. Here, we elaborated the detection principles of CRISPR/Cas12a in in vitro detection. We discussed the different stages leading to the catalytic pathway of target DNA, and the practical applications of Cas12a in nucleic acid detection. These findings revealed a target interference mechanism that originates from the binding of Cas12a-guided RNA complex to complementary DNA sequences within PAM-dependent (dsDNA) regions. The crRNA-DNA binding activates Cas12a, enabling site-specific dsDNA cleavage and non-specific ssDNA trans-cleavage. The release of Cas12a ssDNase activity provides a novel approach to enhance the sensitivity and specificity of molecular diagnostic applications. Before these CRISPR/Cas12a-based nucleic acid detection methods can be introduced into clinical use, substantial work is still required to ensure the accuracy of diagnosis. Nevertheless, we believe that these innovative detection tools based on CRISPR/Cas will revolutionize future diagnostic technologies, particularly offering significant assistance in pathogen infection diagnosis for developing countries with relatively poor healthcare conditions and high prevalence of infectious diseases.

Establishment of national reference panel for coxsackievirus A16 nucleic acid detection kit / 中国生物制品学杂志

@#ObjectiveTo establish the national reference panel for coxsackievirus A16(CA16)nucleic acid detection kit and related quality standard.MethodsThe CA16 positive and negative samples were collected and screened,and then were filled and lyophilized to establish the national reference panel for CA16 nucleic acid detection kit. According to the cooperative calibration results of various reagent manufacturers,the quality standard of reference panel was determined.Meanwhile,the homogeneity and stability of the national reference panel were well studied.ResultsThe national reference panel of CA16 nucleic acid detection kit consisted of 9 positive samples,8 negative samples,1 limit-detecting sample and1 precision sample. The quality standard was as follows:the coincidence rate of positive samples was no less than 8/9;The coincidence rate of negative samples was 8/8;The minimum detection limit required that the dilution of limit-detecting sample was no less than 1∶103;The precision required that the coefficient of variation(CV)of Ct value of 10 precision samples diluted 100 times was no higher than 5% and the results were all positive. The homogeneity of the reference panel met the requirement,and the stability was not affected by the storage at room temperature(25 ℃)for 24 hours and repeated freezing and thawing three times.ConclusionThe first national reference panel of CA16 nucleic acid detection kit and the related quality standard have been established,which provided a reference for the quality control and evaluation of the related reagents.

Influencing factors of negative conversion time of nucleic acid detection in 228 patients infected with Omicron variant of SARS-CoV-2 in Shanghai / 中国热带医学

@#Abstract: Objective To analyze and compare the effects of different clinical characteristics on the negative conversion time of nucleic acid detection after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant infection, and to provide a scientific basis for the isolation and treatment of coronavirus disease 2019 (COVID-19). Methods　The epidemiological and clinical data of 228 mild SARS-CoV-2 Omicron variant infected patients diagnosed in Shanghai were retrospectively collected from April 27, 2022 to June 8, 2022 in Wujiaochang designated Hospital, Yangpu District, Shanghai. The negative conversion time of nucleic acid detection was used as the outcome variable, and the patients were divided into A (≤18 days) and B (>18 days). Univariate and multivariate logistic regression analysis were used to analyze the influencing factors of the negative conversion time of nucleic acid detection. Results　The mean nucleic acid conversion time of 228 patients was (18.7±12.1) d, with the median time of 18 (2-46) d. Among them, 120 patients in group A had an average nucleic acid conversion time of (13.2±2.0) d, and 108 cases in group B had an average nucleic acid conversion time of (20.8±1.3) d. Univariate analysis showed that there were no statistically significant differences in the effects of hypertension, coronary heart disease, diabetes, hypokalemia, malignant tumors, neuropsychiatric diseases, chronic digestive diseases on the negative nucleic acid conversion time (P>0.05); however, there were significant differences in the effects of combined cerebrovascular disease, leukopenia, chronic respiratory system diseases and vaccination on the negative nucleic acid conversion time (P<0.05). Further multivariate logistic regression analysis revealed that the combination of chronic respiratory diseases and non-vaccination were significant risk factors for prolongation of negative nucleic acid conversion time (P<0.05). Conclusions The results of this study show that gender, age and whether hypertension, coronary heart disease, diabetes mellitus, hypokalemia, malignant tumor, neuropsychiatric disease and chronic digestive disease have no significant effect on the nucleic acid conversion time, whereas chronic respiratory disease and no vaccination are significantly correlated with the prolongation of nucleic acid conversion time in SARS-CoV-2 Omicron-infected patients.

Establishment and evaluation of a LAMP-CRISPR-based nucleic acid detection method for Schistosoma japonicum / 中国热带医学

@#Abstract: Objective To establish a sensitive and specific nucleic acid detection method for Schistosoma japonicum based on loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) technology. Methods The LAMP primers, gRNA and ssDNA probe that target Schistosoma japonicum SjR2 genes were designed according to the principles of LAMP and CRISPR. The LAMP-CRISPR reaction system was established and optimized. The sensitivity and specificity of the method were evaluated against the ten-fold serial dilutions of plasmid containing SjR2 target sequences, as well as genomic DNA at different stages of Schistosoma japonicum and other parasites, including Fasciola hepatica, Schistosoma mansoni, Taenia saginata, Clonorchis sinensis, Ascaris lumbricoides, Necator americanus, Paragonimus westermani, and Echinococcus granulosus. Additionally, 15 schistosome-infected snail and 30 uninfected samples were tested by LAMP-CRISPR and LAMP methods, respectively, to evaluate the potential of this method for screening for infected snails. Results　The developed LAMP-CRISPR method was able to specifically amplify and detect the SjR2 gene of S. japonicum. The optimal reaction temperature was 37 ℃, and the optimal reaction concentrations were both 40 nmol/L for gRNA and Cas12a protein. No cross-reaction was observed with genomic DNA from other parasites such as F. hepatica. The detection limit of the method was 10 copies/μL when testing 10-fold dilutions of recombinant plasmids as a template. Furthermore, the LAMP-CRISPR method was able to accurately detect genomic DNA from S. japonicum at various stages of development, including eggs, cercariae, schistosomula, juvenile worms, and adult worms. The results of testing 45 snail samples showed no significant difference between the LAMP-CRISPR and LAMP methods for detecting infected snails (χ2=0.05, P>0.05). The sensitivity and specificity of the LAMP-CRISPR method were 100.00% (15/15) and 96.67% (29/30), respectively, compared to the gold standard, while the sensitivity and specificity of the LAMP method were 100.00% (15/15) and 93.33% (28/30), respectively. Conclusions　This established LAMP-CRISPR detection method presented good sensitivity, specificity and reliability, making it a promising tool for rapid detection and risk monitoring of S. japonicum.

CRISPR-based molecular diagnostics: a review / 生物工程学报

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.

Application of CRISPR/Cas systems in the nucleic acid detection of pathogens: a review / 中国血吸虫病防治杂志

Rapid, sensitive and specific detection tools are critical for the prevention and control of infectious diseases. The in vitro nucleic acid amplification assays, including polymerase chain reaction and isothermal amplification technology, have been widely used for the detection of pathogens. Recently, nucleic acid detection-based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) have been developed, which are rapid, highly sensitive, highly specific, and portable. This review describes the classification and principle of CRISPR/Cas systems and their applications in pathogen detection, and discusses the prospects of CRISPR/Cas systems.

Design and Development of Cloud Platform of Emergency COVID-19 Nucleic Acid Detection / 中国医疗器械杂志

According to the characteristics of short time and large amount of samples for out of hospital emergency nucleic acid detection, this study introduces an out of hospital emergency nucleic acid detection cloud platform system, which realizes the functions of rapid identification of the detected person and one-to-one correspondence with the samples, and real-time upload of the detection results to Zhejiang Government service network for quick viewing and statistics, so as to complete the task of national nucleic acid screening efficiently and accurately that we must provide information support.

Research progress of new isothermal amplification technology in rapid diagnosis of Plasmodium and other parasites / 中国热带医学

@#Abstract: At present, nucleic acid detection technology based on the PCR principle is commonly used to detect malaria parasites, the existing Plasmodium detection methods mainly include microscopy, antigen immunoassay, and nucleic acid detection,but due to the long detection time, high personnel and equipment requirements, and other shortcomings, its popularization, and application at the grassroots level are limited. What challenges previous Plasmodium detection methods are the lack of experienced professionals and advanced equipment at the grassroots as well as the requirement of rapid detection of large samples under extreme conditions. The isothermal amplification technology developed in recent years has potential application prospects due to its simplicity, rapidity, high sensitivity, and high specificity. This article attempts to review the principles, characteristics, and prospects of various isothermal amplification technologies, and on this basis, focuses on the introduction of recombinase polymerase amplification (RPA) and recombinase⁃aided isothermal amplification (RAA) assay technologies and proposes the use of such recombinant enzyme amplification technologies to achieve rapid and accurate diagnosis of common Plasmodium species possibility and imagination.

Efficacy of different detection methods and epidemiological of influenza among preschool children in Leshan City in 2016-2018 / 公共卫生与预防医学

Objective To analyze the epidemiological characteristics of influenza in preschool children in Leshan City from 2016 to 2018, and to compare the difference between antigen colloidal gold reagent and nucleic acid detection reagent. Methods A total of 1 100 patients with suspected influenza admitted to Leshan City from January 2016 to December 2018 were selected as the research objects; the clinical data and characteristics of influenza epidemiology of the patients were collected, and the antigen colloidal gold reagent and nucleic acid detection reagent were used to detect influenza. Epidemiological characteristics; using nucleic acid detection as the standard to evaluate the diagnostic value of antigen colloidal gold reagent detection methods. Results The nucleic acid detection gold standard, antigen colloidal gold reagent method to detect influenza The sensitivity and specificity of the pathogen are both greater than 80%, and the AUC of influenza pathogen detection by the antigen colloidal gold reagent method is greater than 0.90, which has high application value . From 2016 to 2018, Leshan City was dominated by type B Y and type A H3N2; the main clinical features were cough, runny nose, and fever (P<0.05); the distribution of characteristics showed that there was no statistics on the sex of children with influenza Significantly, children less than 5 years old accounted for the highest proportion, and the proportion of children in winter and Spring Festival was significantly higher than that in summer and autumn, and the difference was statistically significant (P<0.05). Conclusion From 2016 to 2018, influenza virus infections in preschool children in Leshan City were mainly type Y and type A H3N2, and showed obvious age and seasonal characteristics. The antigenic colloidal gold reagent was used in clinical screening for children, but the test was negative. Children who are at high risk should be diagnosed with nucleic acid method as soon as possible.

Clinical application of multiplex nucleic acid detection technologies: advancements and future / 中华检验医学杂志

With the advantage of being capable of detecting multiple targets at the same time, high throughput and cost-effective, multiplex nucleic acid detection technologies meet the need of large-scale nucleic acid screening and quantification. Multiplex polymerase chain reaction has been applied to detect pathogen, methylated DNA, mutated gene, and single nucleotide polymorphism typing. Isothermal amplification technologies, such as loop-mediated isothermal amplification and recombinase polymerase amplification are promising in the field of point-of-care testing. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas)-based multiplex nucleic acid detection technologies have become a hotspot due to their high sensitivity and specificity. Metagenomics sequencing plays a leading role in the detection of emerging pathogens and their gene mutation monitoring as well as tumor research. In this review, the advancements and future of multiplex acid detection technologies in clinical application are discussed.

HBV serologic and virus characteristics of HBsAg negative and NAT non-discriminated samples from blood donors / 中国输血杂志

【Objective】 To investigate the HBV infection of TMA initially reactive but discriminatory test non-reactive samples(NDR) after the individual donation nucleic acid detection(ID-NAT)of TMA, and analyze its serological and molecular biological characteristics, so as to improve the safety of blood transfusion. 【Methods】 121 970 samples of blood donors in the center from January 1, 2021 to December 31, 2021 were routinely tested by serology and nucleic acid of ID NAT, and 21 HBsAg(-)/ NDR samples were random collected. After the plasma samples were concentrated by ultra-high speed centrifugation, the gene sequences of BCP/PC, pre-S/S and S region were amplified by Nested PCR. The S region sequence was also sequenced to analyze the viral genotype and amino acid variation. At the same time, the original TMA retest discriminatory test was adopted, and Roche MPX 2.0 was used for ID-NAT, and the samples was not virus-concentrated.NDR samples were supplemented with electrochemiluminescence for anti-HBc and anti-HBs quantitative detection. 【Results】 Of the 121 970 samples screened, 117(0.096%) were found to be HBsAg(-)/NDR samples, of which 21 samples underwent a confirmation test. Sixteen(76.2%) cases were positive for HBV DNA by TMA retest, 7(33.3%) positive for HBV DNA by Roche MPX 2.0 ID-NAT, 9(42.9%) confirmed by Nested PCR, and 8(38.1%) positive by any two methods. Test results of serological markers were as follows: 17(80.9%) positive anti-HBc and 8(38.1%) positive anti-HBs. Eight infected cases were confirmed to have occult hepatitis B infection(OBI). The gene sequence of S region was successfully amplified and sequenced in 3 cases, all of which belonged to C type. Two mutations occurred in specimen S-2, all of which were outside MHR. There were 13 mutations in sample S-6, 6 mutations outside MHR and 7 mutations inside MHR. 【Conclusion】 Nearly 40% of NDR samples can still be detected as HBV DNA positive after virus concentration. Anti-HBc has a high detection rate, and there may be a potential risk of HBV transmission. The current NAT detection sensitivity should be improved. The amino acid mutation of S gene sequence may be related to OBI formation.

Long-term immunization and prognosis outcome of patients with occult hepatitis B virus infection / 中国输血杂志

【Objective】 To investigate the prognosis of blood donors with occult hepatitis B virus infection (OBI) by long-term follow-up and repeated testing of HBsAg and HBV DNA. 【Methods】 From January 1, 2010 to December 31, 2020, voluntary blood donors were screened by both serological and viral nucleic acid(NAT) testing, then samples were further confirmed as HBV DNA positive by manual nested-PCR amplification.A total of 306 cases were detected as HBsAg negative /HBV DNA positive, then followed-up for a long time and re-examined of HBsAg and HBV DNA to confirm whether they had infected with OBI.The prognosis of patients with OBI who experienced long-term immunization was determined by repeated testing. 【Results】 A total of 306 HBsAg negative/ HBV DNA positive blood donors had been followed up, and 40(13.07%, 40/306) were recalled frequently for re-examination.Among them, 90%(36/40), 57.5%(23/40), 40% (16/40)were anti-HBc + , anti-HBs + and anti-HBe + , respectively, and 50%(20/40), 40%(16/40), 7.5%(3/40) and 2.5% (1/40)were anti-HBs+ / anti-HBc + , anti-HBc + / anti-HBs -, anti-HBc -/ anti-HBs + and anti-HBc -/ anti-HBs -, respectively.Those 40 blood donors were followed-up for 1-13 times, with the duration of 8-108 months (0.6~9 years).1 donor (2.5%) was followed-up less than 1 year, 11 (27.5%)>1 year and ≤3 years, 23 (57.5%) 23(57.5%)>3 years and ≤5 years, and 5 (12.5%) for more than 5 years.After long-term following up and repeated testing, 50%(20/40)of OBI blood donors turned negative for HBV DNA (HBsAg negative / HBV DNA negative), 42.5% (17/40)were confirmed as OBI infection (HBsAg negative / HBV DNA positive), and 7.5%(3/40) were hard to determine (after repeated testing, the results were either positive or negative). 【Conclusion】 After long-term following up and repeated screening, we found that none of the OBI patients turned into acute or chronic HBV infection, and most of them maintained OBI.However, OBI blood donors carry very low load of HBV DNA for a long time, which could lead to false negative results of NAT and bring a great challenge to the safety of blood transfusion.

Classification management of HIV reactive blood donors based on test results / 中国输血杂志

【Objective】 To explore the viability of classification management of HIV reactive blood donors based on test results in blood screening laboratory. 【Methods】 According to the HIV test results of blood donors (including twice ELISA and once NAT), the HIV reactive blood donors were divided into three groups. Group 1 was all-test reactive (both ELISA and NAT were reactive), group 2 serological reactive (only ELISA was reactive), and group 3 NAT reactive (only NAT was reactive). The HIV test results of 191 628 blood donors from May to December 2017 were analyzed. Samples with positive RIBA results and / or the repeated reactive NAT results were determined as HIV true positive. The yielding rates of HIV true positivity in each group were analyzed. Receiver operating characteristic curve (ROC curve) was used to elevate the S/CO limit under 99% specificity as the blood donor deferral limit for ELISA. 【Results】 A total of 180 HIV reactive samples were detected out of 191 628 blood donors, including 77 positive cases in group 1, 100 in group 2 and 3 in group 3. 1) The HIV reactive results were diverse. Among the 82 true positive blood donors, 4 were early HIV infection (3 HIV antibody+ antigen window period yield, 1 HIV antibody window period yield), 2 were suspected elite controllers, and 76 cases were both serology and NAT reactive. 2) The overall yielding rate of HIV was 47.67%, with group 1 (100%) = group 3 (100%) > group 2 (2.17%), showing statistically significant (P0.05). All true positive blood donors in group 1 and group 2 could be accurately screened by using the blood donor deferral limit for ELISA1 and ELISA2 simultaneously. 【Conclusion】 The composition of HIV results among blood donors is diverse and complex. It is necessary to continuously improve the awareness of HIV prevention and control. The classification of HIV reactive blood donors is conducive to conduct fine and scientific management. The blood donors in group 1 and group 3 should be permanently deferral, and the suspected HIV elite controllers in group 2 should be paid attention to and permanently deferral.

Comparison of disinfection effects of different methods for nucleic acid pollution in 2019-nCoV / 西安交通大学学报(医学版)

【Objective】 To evaluate the ability of different disinfection methods to remove nucleic acid pollution in 2019-nCoV so as to obtain the best removal scheme. 【Methods】 2019-nCoV positive quality control nucleic acid of 50 μL was applied to plastic， metal and glass with medical cotton swabs， respectively. After drying， we dropped 50 μL of 750 mL/L alcohol （ethanol）， chlorine-containing disinfectant （2 000 mg/L and 5 500 mg/L）， and PCR Cleaner， respectively. After 1 min， the contaminated area was wiped with medical cotton swabs and soaked in 300 μL of pure water. After shaking and mixing， 5 μL was taken as a template. The Ct values of ORF1ab and N genes and IC genes of internal standard fragment in the amplified target area of 2019-nCoV after wiping with different disinfection methods were compared to evaluate the effect of eliminating nucleic acid pollution， and each experiment was repeated for three times. Similarly， the effects of ultraviolet irradiation for 0， 1， 2， 3， 4， and 5 hours on the removal of nucleic acid pollution were compared. 【Results】 After 2 000 mg/L and 5 500 mg/L chlorine-containing disinfectant wiped the contaminated area， the Ct values of ORF1ab and N genes and IC genes of internal standard fragment in the amplified target area in 2019-nCoV were all 0， and the Ct values of all genes in the contaminated area in groups 3， 4 and 5 h after UV irradiation were all 0， which completely cleared the pollution and had a strong effect. The effect of PCR Cleaner was second， and 750 mL/L ethanol was the worst. 【Conclusion】 2 000 mg/L and 5 500 mg/L chlorine-containing disinfectant and ultraviolet irradiation for 3 hours have the best effect of eliminating nucleic acid pollution， which is worth popularizing under appropriate conditions.

Performance evaluation of six coronavirus nucleic acid detection reagents and matching analysis with nucleic acid extraction reagents / 中华检验医学杂志

Objective:To select nucleic acid extraction reagents and amplification reagents by comparing the minimum detection limit, amplification efficiency, specificity, accuracy, and matching analysis with 6 nucleic acid extraction reagents of 6 novel coronavirus (2019-nCoV) nucleic acid detection reagents and provide scientific basis for clinical diagnosis and treatment.Methods:The national-standard material of 2019-nCoV nuclear acids was diluted as ths six fradient concentrations. Then, the diluted samples were analyzed by using the six kinds of agents/kits, Guangzhou Daan (DA), Beijing Jinhao (JH), Wuhan Mingde (MD), Sichuan Mike (MK), Jiangsu Shuoshi (SS) and Shanghai Zhijiang (ZJ). The minimum detection limit, amplification efficiency, accuracy, specificity and other properties of these kits were examined. The positive control levels 1, 2 (low and medium concentration) were extracted by using the six nucleic acid extraction reagents (numbered a-f) with six 2019-nCoV detective kits to detect target genes of 2019-nCoV genome, and compared between different nucleic acid extraction reagents and different nucleic acid detection reagents. Match between the different nucleic acid extraction reagents detection reagents, using two-way analysis of variance.Results:The minimum detection limit of Nucleocapsid (N) gene of DA, JH, MD, SS reagent was 7.0×10 2 copies/ml.The minimum detection limit of open reading frame 1ab (ORF1ab) gene of JH, MD, SS, MK reagent was 9.39 ×10 2 copies/ml.The minimum detection limit of envelope (E) gene of MK, ZJ reagent was 5.03×10 2 copies/ml. SS reagent N gene amplification efficiency was 89%, ORF1ab gene amplification efficiency was 90%, was the highest among six reagents.a, b, c, d extraction reagents and 6 nucleic acid detection reagents match well, But a extraction reagent was more suitable for use with SS,MK and ZJ reagents ( P<0.05), d extraction reagent was more suitable for MD,SS,DA,JH and MK nucleic acid detection reagents( P<0.05); e extraction reagent was more suitable for SS nucleic acid detection reagents( P<0.05), f extraction reagent was most suitable for DA, JH Nucleic acid detection reagents ( P<0.05); e and f reagents were not suitable for use with MK and ZJ. Conclusion:The 6 detection reagents have good performance, and the appropriate extraction reagents and detection reagents should be used in conjunction with the actual conditions of the laboratory.

A novel test strip assay based on gold nanoparticles-labeled nucleic acid probe for rapid detection of Vibrio splendidus / 生物工程学报

Vibrio splendidus is an opportunistic pathogen in aquaculture. It can infect a variety of aquaculture animals and has caused huge losses to the aquaculture industry. In this study, a novel and efficient method for detecting V. splendidus was developed by combining the exonuclease Ⅲ amplification strategy with a nucleic acid test strip developed based on gold nanoparticles-labeled DNA probe. The results could be directly visualized by naked eyes, and this system overcame the difficulty in preparation of the monoclonal antibody used in conventional immunostrip. Upon optimization of experimental conditions, the detection limit of the strip was 5 ng/mL for the synthetic oligonucleotide DNA fragment and 10 ng/mL for the actual genomic DNA sample of V. splendidus. This test strip was more sensitive compared with the PCR method and was specific for the detection of V. splendidus. The rapid preparation of nucleic acid strip and the efficient detection of V. splendidus open a new way for the prevention and control of aquatic diseases.

Application of CRISPR/Cas-based biosensors for detecting nucleic acid of pathogens / 生物工程学报

Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats -associated protein (CRISPR/Cas) has been developed as a precise, efficient, affordable and sensitive nucleic acid detection tool due to its efficient targeted binding ability and programmability. At present, biosensors based on CRISPR-Cas system have shown excellent performance in the detection of nucleic acid of pathogens, which has attracted widespread attention, and is expected to replace the conventional detection methods. This review summarizes the latest research progress of biosensors based on CRISPR/Cas system for detecting nucleic acid of pathogens.

Establishment of a nucleic acid dipstick test for detection of Schistosoma japonicum specific gene fragments based on the recombinase-aided isothermal amplification assay / 中国血吸虫病防治杂志

Objective To develop a rapid test for detection of Schistosoma japonicum specific gene fragments based on the recombinase-aided isothermal amplification assay (RAA) and nucleic acid dipstick test. Methods The S. japonicum SjG28 gene fragment was selected as the target gene fragment, and the primers and fluorescent probe were designed and synthesized. Then, a S. japonicum nucleic acid dipstick test was established. The sensitivity of this dipstick test was evaluated by detecting different copies of recombinant plasmids containing the S. japonicum SjG28 gene fragment and different concentrations of genomic DNA from adult worms of S. japonicum, and the specificity of the dipstick test was evaluated by detecting the genomic DNA from Clonorchis sinensis, S. mansoni, Ancylostoma duodenale, S. haematobium, Babesia and Paragonimus westermani. Results The S. japonicum nucleic acid dipstick test based on the S. japonicum SjG28 gene fragment showed the minimum detectable limit of 10 copies/μL of the recombinant plasmid containing the S. japonicum SjG28 gene fragment and the minimum detectable limit of 1 pg/μL of S. japonicum genomic DNA, and the dipstick assay tested negative for the genomic DNA from C. sinensis, S. mansoni, A. duodenale, S. haematobium, Babesia and P. westermani. Conclusion A rapid, simple, and visualized assay is established for detection of S. japonicum specific gene fragments based on RAA and nucleic acid dipstick test.