BIOTECHNOLOGY AND BIOENGINEERING
·
2021
·Vol. 118
·期 5
·2053-2066
·
IF 4.53
Wei Lin,Tian Tian,Yongzhong Jiang,Erhu Xiong,Debin Zhu,Xiaoming Zhou
Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.
研究领域:
分子生物学
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医学诊断
·
基因编辑
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DOI
PubMed
高影响因子
ANTIVIRAL RESEARCH
·
2014
·
IF 3.434
Long Yin,Yuzi Luo,Bo Liang,Fei Wang,Min Du,Valery A. Petrenko,Hua-Ji Qiu,Aihua Liu
Classical swine fever (CSF) is a devastating infectious disease caused by classical swine fever virus (CSFV). The screening of CSFV-specific ligands is of great significance for diagnosis and treatment of CSF. Affinity selection from random peptide libraries is an efficient approach to discover ligands with high stability and specificity. Here, we screened phage ligands for the CSFV E2 protein from f8/8 landscape phage display library by biopanning and obtained four phage clones specific for the E2 protein of CSFV. Viral blocking assays indicated that the phage clone displaying the octapeptide sequence DRATSSNA remarkably inhibited the CSFV replication in PK-15 cells at a titer of 10 10 transduction units, as evidenced by significantly decreased viral RNA copies and viral titers. The phage-displayed E2-binding peptides have the potential to be developed as antivirals for CSF.
研究领域:
病毒学
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分子生物学
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医学
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产品关联
DOI
PubMed
高影响因子
ARCHIVES OF VIROLOGY
·
2021
·Vol. 166
·期 3
·871-879
·
IF 2.574
Zhang Xinyu,Liu Xiaoyu,Wu Xiaodong,Ren Weijie,Zou Yanli,Xia Xiaoli,Sun Huaichang
African swine fever (ASF), caused by African swine fever virus (ASFV), was first reported in Kenya in 1921, but an effective vaccine or antiviral drug is still not available for ASFV control. Rapid and effective diagnostics are key steps in managing ASF. We generated two monoclonal antibodies (MAbs) against the ASFV phosphoprotein P30 and designated these as 3H7A7 and 6H9A10. Epitope mapping revealed that MAb 3H7A7 and 6H9A10 recognized aa 144-154 and aa 12-18 of P30, respectively. A signal-amplified sandwich colloidal gold test strip for rapid detection of ASFV was developed based using these MAbs. Sensitivity and specificity analysis showed that the detection limit of the strip was 2.16 ng of P30. The strip only reacted with ASFV and did not react with other common porcine viruses. In detection tests using 153 clinical field samples including sera, plasma, anticoagulant-treated blood, and tissue, the strip had 95.42% concordance with real-time PCR. The new MAbs specific for P30 and the rapid colloidal gold test strip helped to reveal novel B cell epitopes in P30 and provide an efficient diagnostic test for on-site clinical detection of ASF.
研究领域:
兽医学
·
分子生物学
·
免疫学
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产品关联
DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2021
·Vol. 105
·期 11
·4751-4759
·
IF 4.813
Chen Dongjie,Wang Di,Wang Caixia,Wei Fang,Zhao Hongyuan,Lin Xiangmei,Wu Shaoqiang
Abstract Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and “sandwiched” by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78–100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. Key points • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.
研究领域:
兽医学
·
分子诊断学
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免疫学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Jingming Zhou,Wan Yu,Xifang Zhu,Hongliang Liu,DongMin Liu,Aiping Wang,Gaiping Zhang
ASFV is the only known double-stranded insect-borne DNA virus, which can rapidly infect domestic pigs and wild boars with ticks as transmission medium. Since it was first discovered in 1921, it quickly spread to all parts of the world and brought huge economic losses to the pig industry all over the world. At present, there is still no safe and effective vaccine for ASFV. Here, we developed a quantum-dot labeled antibody test strip for the detection of antibodies against ASFV pp62. The pp62 protein was labeled with quantum dots, and the antibody test strip was developed uses it in a detection mode of labeled antigen-SPA interceptor-monoclonal antibody quality control. The test strip showed high sensitivity, the positive detection limit of the strip was 1: 10 6 by continuous multiple dilution using the positive standard serum of ASFV antibody as reference. The test strip showed good specificity, and there was no cross reaction with other swine diseases virus (PCV2, PRRSV, CSFV, PPV). Using the detection results of commercialized kit for African swine fever virus as reference, 80 ASFV antibody negative serum and 4 different ASFV antibody positive serum were detected using the ASFV pp62 quantum-dot labeled antibody test strip. The results were consistent with the commercial kit. This study provides a new detection method for the prevention and control of African swine fever.
研究领域:
兽医学
·
分子生物学
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诊断学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
AMB Express
·
2025
·Vol. 15
·期 1
·1-12
·
IF 3.7
Hu Yanli,Xia Yingjun,Mo Huiwen,Zhang Li,Cao Wenjian,Fang Rui,Zhao Junlong
African swine fever (ASF) is a contagious viral disease in pigs, caused by the African swine fever virus (ASFV). Currently, there are no effective vaccines and drugs for ASFV, making diagnostic methods crucial for the prevention and control of ASF. In this study, the ASFV p30 protein was successfully expressed using a prokaryotic expression system and used as an immunogen to prepare monoclonal antibody (mAb) 2A5. The antigenic epitope recognized by the mAb 2A5 was identified as 58 VKYDIVKSARIYAGQGY 74 . Then, a blue latex microsphere immunochromatographic strip method for detecting ASFV antigens was established. Wherein a rabbit polyclonal antibody (pAb) against p30 stored in our laboratory was coupled with blue latex microspheres to prepare an immune probe, with mAb 2A5 as the test line and goat anti-rabbit IgG as the control line. The immunochromatographic strip exhibited excellent sensitivity, with a minimum detection limit of 10 ng/mL for the p30 protein. It demonstrated good specificity, showing no cross-reactivity with PRRSV, PCV2, PPV, and PEDV. Moreover, it maintained good stability and could be stored for at least 100 days at 37 °C. The concordance rate between the immunochromatographic strip and qPCR test kit was 92.2%. The immunochromatographic strip method can meet the requirements of clinical testing and provide technical support for ASFV diagnosis.
研究领域:
兽医学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2022
·
IF 6.556
Siwenjie Qian,Yanju Chen,Cheng Peng,Xiaofu Wang,Hui Wu,Yang Che,Huanying Wang,Junfeng Xu,Jian Wu
African swine fever virus (ASFV) can cause highly contagious and fatal disease among domestic pigs, resulting in considerable economic losses for swine breeders. There is a strong demand for accurate, rapid, and simple detection methods especially for on-site application. Nucleic acid testing is the most commonly used method for ASFVdetection. However, traditional nucleic acid purification step is time- and labor-consuming. The nucleic acid purification, amplification and amplicons detection rely on laboratory settings which limits the on-site detection. Here, we proposed a simple and cost-effective detection method that utilized filter paper to purify nucleic acids from swine blood and employed CRISPR/Cas12a-mediated loop-mediated isothermal amplification (LAMP) reaction to detect ASFV. The filter paper which was made into dipsticks could effectively purify nucleic acids from whole blood in 2 min. This simple and low-cost purification method avoided multiple pipetting steps and potential amplification inhibitors (e.g., ethanol) that were generally used in traditional nucleic acids extraction processes. After nucleic acid purification, the lyophilized LAMP reagent dissolved by elution solution was employed to perform isothermal amplification reaction on a portable heating block. The CRISPR/Cas12a system was designed to specifically detect amplicons. Assisted by a portable homemade device, the fluorescent signals produced by positive samples could be observed by the naked eye, while negative samples remained colorless. The whole detection procedure could be finished within 50 min with a detection limit of one copies/μL. This established method provided a novel strategy for rapid visualized detection and showed great potential for on-site application.
研究领域:
兽医学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2025
·
IF 6.1
Yuhao Huang,Huihui Li,Jian Wen,Shaowen Li,Qigai He,Xianrong Meng
Currently, antigen and antibody early detection is an important part in the control of African swine fever. However, traditional detection technologies based on antigen-antibody specific binding face problems such as low sensitivity, difficulty in preparing antibodies, and significant differences between batches of antibodies, which require the search for a new type of diagnostic molecule and detection technology. This study employed magnetic beads-SELEX to identify aptamers that specifically bind to the African swine fever virus (ASFV) p30 protein. The aptamer (p30-1) has high affinity and specificity, with no cross-reactivity to His-tagged or GST tagged proteins. In addition, the thiol-modified aptamer was used to immobilize ASFV p30 protein onto an electrochemical immunosensor constructed with thiol-modified multi-walled carbon nanotubes (S-MWCNTs) and gold nanoparticles (AuNPs). The detection of ASFV p30 monoclonal antibody was carried out using differential pulse voltammetry (DPV), and a standard curve was established. The linear detection range was 0.1 μg/mL-4 μg/mL, with a limit of detection (LOD) of 1.38 μg/mL. This method shows good specificity and reproducibility, enabling effective detection of clinical serum samples and infected animal serum samples. It provides new technical support for the rapid detection of ASFV antibodies.
研究领域:
兽医学
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分子生物学
·
生物传感技术
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产品关联
DOI
PubMed
高影响因子
Viruses-Basel
·
2024
·Vol. 16
·期 3
·449
·
IF 3.8
Junfei Dai,Xusheng Ma,Ashenafi Kiros Wubshet,Qian Li,Xiaofen Shang,Zhikuan Luo,Jianan Liu,Zhiyu Li,Mingxia Li,Yujie Song,Lijun Guo,Jie Zhang,Haixue Zheng
African swine fever (ASF) is a highly contagious and hemorrhagic disease caused by infection with the African swine fever virus (ASFV), resulting in a mortality rate of up to 100%. Currently, there are no effective treatments and commercially available vaccines for ASF. Therefore, it is crucial to identify biochemicals derived from host cells that can impede ASFV replication, with the aim of preventing and controlling ASF. The ASFV is an acellular organism that promotes self-replication by hijacking the metabolic machinery and biochemical resources of host cells. ASFV specifically alters the utilization of glucose and glutamine, which are the primary metabolic sources in mammalian cells. This study aimed to investigate the impact of glucose and glutamine metabolic dynamics on the rate of ASFV replication. Our findings demonstrate that ASFV infection favors using glutamine as a metabolic fuel to facilitate self-replication. ASFV replication can be substantially inhibited by blocking glutamine metabolism. The metabolomics analysis of the host cell after late-stage ASFV infection revealed a significant disruption of normal glutamine metabolic pathways due to the abundant expression of PLA (phenyllactic acid). Pretreatment with PLA also inhibited ASFV proliferation and glutamine consumption following infection. The metabolomic analysis also showed that PLA pretreatment greatly slowed down the metabolism of amino acids and nucleotides that depend on glutamine. The depletion of these building blocks directly hindered the replication of ASFV by decreasing the biosynthetic precursors produced during the replication of ASFV’s progeny virus. These findings provide valuable insight into the possibility of pursuing the development of antiviral drugs against ASFV that selectively target metabolic pathways.
研究领域:
病毒学
·
代谢生物学
·
动物健康
·
药理学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2021
·
IF 6.558
Jiahao Li,Yun Bai,Fang Li,Yue Zhang,Qingyun Xie,Lei Zhang,Lizhong Hua,Qiyan Xiong,Yanke Shan,Zhigao Bu,Guoqing Shao,Zhixin Feng,Dongming Zhao,Fei Liu
African swine fever (ASF) is a swine viral disease that could cause highly contagious and extremely high mortality, causing huge economic losses to the pig industry. As there is currently no vaccine and effective treatment methods. Therefore, early monitoring is one of the most important solutions to prevent and control ASF. In this study, the dual QDM recombinant virus protein 30 and 54 (P30 and P54) probes and pre-incubation in vitro were proposed for the first time as QDM based-ASFV immunosensor (QAIS) for the ultra-sensitive quantitative detection of ASFV antibodies in serum. In the range from serum dilution of 1:1000 to 1:64000, it showed a good linear relationship (R 2 = 0.9947), and its detection sensitivity was 1:64000 dilution. Compared with commercial enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic strip (CGICS), its detection sensitivity was improved by at least one order of magnitude and four orders of magnitude respectively. In addition, the whole ASFV antibody screening test can be completed in 25 min with simple operation. The performance and practicability of the established QAIS sensor have been verified by ASF-ELISA kit, and its coincidence rate was as high as 98.7% in 151 clinical samples. We firmly believe that the proposed QAIS sensor could potentially be applied to point-of-care testing (POCT) for quantitative ASFV antibody in pig farms.
研究领域:
兽医学
·
免疫学
·
诊断技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2022
·Vol. 106
·期 7
·2703-2714
·
IF 5.56
Geng Rui,Sun Yaning,Li Rui,Yang Jifei,Ma Hongfang,Qiao Zixuan,Lu Qingxia,Qiao Songlin,Zhang Gaiping
Abstract African swine fever virus (ASFV) causes a highly contagious and often lethal swine viral disease, and leads to tremendous economic losses to the swine industry. Unfortunately, there are no vaccines and effective antiviral agents available to prevent and control ASFV outbreaks. Therefore, it is necessary to develop simple and rapid strategies to monitor ASFV-infected pigs to restrain its spread. In the current study, ASFV capsid protein p72 was expressed along with its chaperone pB602L to form trimers in human embryonic kidney 293 (HEK293) cells. The p72 trimers were subsequently labeled with colloidal gold to develop a immunochromatographic strip. The strip showed high specificity to ASFV-positive serum and no cross-reactivity to other swine virus positive sera. Importantly, the strip showed a higher sensitivity of detecting ASFV antibodies in both positive standard serum and clinical serum samples than a commercial enzyme-linked immunosorbent assay (ELISA) kit. Taken together, these results demonstrate the strip as a reliable diagnostic tool against ASFV infection, which will be appropriate for application in prevention and control of ASFV.
Key points
• ASFV p72 trimers were successfully generated . • A colloidal gold strip was developed based on ASFV p72 trimers . • The strip is appropriate for detecting ASFV antibodies in the field .
研究领域:
兽医学
·
分子生物学
·
诊断技术
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Guobin Mao,Xing Luo,Silu Ye,Xun Wang,Jin He,Jilie Kong,Junbiao Dai,Wen Yin,Yingxin Ma
It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.
研究领域:
病毒学
·
分子生物学
·
生物技术
·
分析化学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）