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@#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.
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@#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.
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@#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.
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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.
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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.
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Humans , CRISPR-Cas Systems/genetics , COVID-19/genetics , Pandemics , Artificial Intelligence , Nucleic AcidsABSTRACT
【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.
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【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.
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【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.
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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.
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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.
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Humans , COVID-19 , Cloud Computing , Nucleic Acids , SARS-CoV-2ABSTRACT
@#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.
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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.
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Objective To establish a nucleic acid assay for detection of Paragonimus skrjabini based on the recombinase-aided isothermal amplification (RAA) technique, and to preliminarily evaluate its detection efficiency. Methods The metacercariae of P. skrjabini, P. westermani and Euparagonimus cenocopiosus were isolated from crabs, and genomic DNA was extracted for molecular characterization. The cytochrome coxidase 1 (cox1) gene sequence of P. skrjabini was selected as the target gene fragment, and the primers and probes were designed, screened and synthesized for RAA assay. The genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province were used as templates for verification of the fluorescent RAA assay. The fluorescent RAA assay was performed to detect different concentrations of plasmids containing target gene fragment and P. skrjabini metacercariae genomic DNA to determine the sensitivity. Fluorescent RAA assay was performed with recombinant plasmids containing P. skrjabini cox1 gene sequences at different concentrations and P. skrjabini genomic DNA as templates to evaluate its sensitivity, and the genomic DNA of P. westermani, E. cenocopiosus, Clonorchis sinensis and Schistosoma japonicum was detected with fluorescent RAA assay to evaluate its specificity. Results P. skrjabini, P. westermani and E. cenocopiosus metacercariae were isolated from crabs, respectively. Molecular characterization and phylogenetic analysis confirmed their homology with the genes sequences of standard Paragonimus strains in GenBank. A fluorescent RAA assay was successfully established for nucleic acid detection of P. skrjabini, and the genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province was amplified using the fluorescent RAA assay within 5 min, while the negative control was not amplified. If the recombinant plasmid containing P. skrjabini cox1 gene sequences was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 copies/μL, and positive amplification was observed within 5 min. If genomic DNA was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 pg/μL, and all positive amplifications were found within 5 to 10 min. In addition, the fluorescent RAA assay was tested negative for P. westermani, E. cenocopiosus, C. sinensis and S. japonicum. Conclusions A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of P. skrjabini, which has potential values in rapid field detection and species identification in freshwater crabs in areas endemic for P. skrjabini.
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Objective To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Methods Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. Results After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. Conclusions A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.
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Nucleic acid detection technique has good sensitivity and specificity and is widely used in in vitro diagnosis, animal and plant commodity quarantine, forensic identification, and other fields. However, it is susceptible to carryover contamination during the operation and leads to false-positive results, which seriously affects the detection accuracy. Therefore, finding an effective solution to prevent and eliminate nucleic acid carryover contamination has become particularly urgent. This study compared several different methods for removing nucleic acid contamination and confirmed that sodium hypochlorite solution and PCRguard reagent could effectively eliminate nucleic acid carryover in the liquid and on surfaces of different materials. Besides, the combination of sodium hypochlorite solution and PCRguard can solve the nucleic acid aerosol contamination. This study proposes solutions for the routine prevention of carryover contamination and removal of aerosol that has occurred in molecular diagnostic laboratories.
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Laboratories , Nucleic Acids , Pathology, MolecularABSTRACT
【Objective】 To investigate the epidemiology of HTLV by conducting HTLV nucleic acid detection among voluntary blood donors, so as to provide basis for the decision making of blood screening strategy. 【Methods】 The HTLV blood nucleic acid(NAT) screening platform was established based on the existing NAT for HBV / HCV / HIV screening. HTLV (type 1 + 2) detection was carried out in 5 368 blood donors, and the results were analyzed. 【Results】 No NAT-yielding of HTLV- 1, -2 type was found in a total of 5 368 voluntary blood donor from January to August 2019. 【Conclusion】 Qianxinan, currently, is very low epidemic or non epidemic as none of HTLV infections was found among blood donors and no significant differences in the epidemiology of HTLV were notable between the counties and cities. It, however, still needs further investigation in the future.
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【Objective】 To compare the analytical performance of Tigris, Panther, ChiTaS BSS1200 and cobas S201 system to see if they satisfy the requirements of blood screening and to know the concordance of the results presented by these four systems. 【Methods】 According to the relevant documents of ISO15189 and Standard Operating Procedure of Blood Station(2019), the parameters needed to be verified for nucleic acid tests(NAT) included: analytical sensitivity verification, system compare test, anti-jamming capability and anti-cross-contamination ability. 【Results】 The 95% detection limits of Tigris, Panther, ChiTaS BSS1200 and Cobas S201 for HBV-DNA(IU/mL), HIV-RNA(IU/mL) and HCV-RNA(IU/mL) were 2.013 vs 4 vs 2.995 vs 0.99, 13.039 vs 10.21 vs 30.952 vs 32.24, and 2.278 vs 2.077 vs 12.008 vs 3.39, respectively. In the performance comparison verification between NAT systems, the results of the two sets of Tigris systems were in full accordance with serum plate, with a concordance rate of 100%, Kappa value of 1, and none cross-contamination.The concordance rate of No.1 Panther system was 100%, and No.2 98%, with Kappa value of 0.961 and none cross-contamination. Hemolytic samples (5g/L Hb concentration) and lipemic blood samples (13.81 mmol/L TG concentration) had no significant effect on the detection of low-concentration samples. 【Conclusion】 No significant differences in the performance of NAT systems were notable by devices, as the four systems were fully automated with high sensitivity, which can fully satisfy the blood screening requirements. Panther system demonstrates superior analysis sensitivity in HCV-RNA/HIV-RNA and lower in HBV DNA, but also in required criteria, as compared to Tigris system. Neither hemolysis nor lipemic blood had any significant effect on the test results.
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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.
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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 sum-marized useful insights into the molecular diagnosis of COVID-19 under the special situation being challenged by virus mutation and asymptomatic infection.
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Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (