Jove-Journal of Visualized Experiments
·
2019
·
IF 1.108
Yafeng Zhang,Xun Liao,Peihua Luo,Kui Peng,Wenyi Ye,Tengxiang Lian,Qibin Ma,Hai Nian
In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5' triphosphate. However, the triphosphate at primary 5' termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5' polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5' triphosphate.In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.
研究领域:
植物学
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分子生物学
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PubMed
高影响因子
LWT-FOOD SCIENCE AND TECHNOLOGY
·
2024
·
IF 6
Wenwen Zhang,Ye Yang,Fei Kong,Yubin Chen,Zhonghua Wang,Jun Zhou,Xiurong Su,Jiajie Xu
Foodborne infectious diseases, particularly those caused by pathogenic Vibrio species in aquaculture, are a significant concern in the field of seafood safety. They not only hinder the economic development of the industry but also pose a major threat to public health. Traditional detection methods are often too slow, insensitive, or impractical for portable use, highlighting the urgent need for a sensitive, on-site detection method for Vibrio that prioritizes convenience and speed. This study developed a visual IMBS-CPCR method for the detection of four common pathogenic Vibrio species: Vibrio alginolyticus , Vibrio anguillarum , Vibrio fluvialis , and Vibrio harveyi . The method achieved high capture rates for target bacteria (82.6%–83.3%) and low capture rates for non-target bacteria (below 11%), demonstrating good sensitivity by detecting bacteria at concentrations as low as 10 CFU/mL. Optimal amplification conditions were determined for each species, with specificity tests confirming that only target bacteria produced amplification bands. Sensitivity tests revealed detection limits of 10 1 CFU/mL for V. alginolyticus , V. anguillarum , and V. fluvialis , and 10 2 CFU/mL for V. harveyi . The combined techniques improved detection sensitivity in simulated fish samples by 10–100 fold compared to direct CPCR or PCR methods.
研究领域:
微生物学
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分子生物学
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食品科学
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高影响因子
PLoS Neglected Tropical Diseases
·
2020
·Vol. 14
·期 8
·e0008648
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IF 3.885
Jiazhen Zheng,Rui Zhou,Fengjuan Chen,Guofang Tang,Keyi Wu,Furong Li,Huamin Liu,Jianyun Lu,Jiyuan Zhou,Ziying Yang,Yuxin Yuan,Chunliang Lei,Xianbo Wu
The phenomenon of COVID-19 patients tested positive for SARS-CoV-2 after discharge (redetectable as positive, RP) emerged globally. The data of incidence rate and risk factors for RP event and the clinical features of RP patients may provide recommendations for virus containment and cases management for COVID-19. We prospectively collected and analyzed the epidemiological, clinical and virological data from 285 adult patients with COVID-19 and acquired their definite clinical outcome (getting PCR positive or not during post-discharge surveillance). By March 10, 27 (9.5%) discharged patients had tested positive for SARS-CoV-2 in their nasopharyngeal swab after a median duration of 7·0 days (IQR 5·0–8·0). Compared to first admission, RP patients generally had milder clinical symptoms, lower viral load, shorter length of stay and improved pulmonary conditions at readmission (p<0.05). Elder RP patients (≥ 60 years old) were more likely to be symptomatic compared to younger patients (7/8, 87.5% vs . 3/19, 18.8%, p = 0.001) at readmission. Age, sex, epidemiological history, clinical symptoms and underlying diseases were similar between RP and non-RP patients (p>0.05). A prolonged duration of viral shedding (>10 days) during the first hospitalization [adjusted odds ratio [aOR]: 5.82, 95% confidence interval [CI]: 2.50–13.57 for N gene; aOR: 9.64, 95% CI: 3.91–23.73 for ORF gene] and higher Ct value (ORF) in the third week of the first hospitalization (aOR: 0.69; 95% CI: 0.50–0.95) were associated with RP events. In conclusion, RP events occurred in nearly 10% of COVID-19 patients shortly after the negative tests, were not associated with worsening symptoms and unlikely reflect reinfection. Patients’ lack of efficiency in virus clearance was a risk factor for RP result. It is noteworthy that elder RP patients (≥ 60 years old) were more susceptible to clinical symptoms at readmission.
The baseline enrolled 285 patients admitted to Guangzhou Eighth People’s Hospital (Guangzhou, Guangdong) with a diagnosis of COVID-19. We reported the epidemiology, clinical laboratory, radiological characteristics, virological results, treatment, and definite outcomes (getting PCR retested positive (RP) or not during post-discharge surveillance) of the cases. RP events occurred in nearly 10% of cases, were not associated with worsening symptoms and unlikely reflect reinfection. The lack of efficiency in virus clearance was a risk factor for RP result. Elder RP patients (≥ 60 years old) were more susceptible to clinical symptom at readmission. In the context of numerous COVID-19 cases showed SARS-CoV-2 positive again after discharged, the data in China may provide recommendations for post-discharge management, especially for other developing countries.
研究领域:
流行病学
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传染病学
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公共卫生
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PubMed
高影响因子
MICROCHIMICA ACTA
·
2025
·Vol. 192
·期 4
·1-11
·
IF 5.3
Han Lu,Song Yaqi,Chen Min,Pan Leiqing,Tu Kang,Su Jing
An upconversion-gold nanoparticle detection system that integrates PCR amplification and fluorescence resonance energy transfer was constructed to enable swift and highly sensitive identification of Escherichia coli . The forward primer used in the PCR amplification is modified with sulfhydryl groups, enabling its connection to gold nanoparticles via Au–S bonds. The complementary strand of the forward primer, which is attached to the upconversion nanomaterials, can hybridize with the free forward primer through base complementary pairing. This interaction induces fluorescence resonance energy transfer, resulting in fluorescence quenching. The concentration of the target bacteria influences the amount of free primer in the system after PCR amplification, which subsequently alters the intensity of the upconversion fluorescence. The fluorescent PCR sensor developed based on the aforementioned principles demonstrated a detection limit of 14 CFU/mL for E. coli , with a quantitative detection range of 18–1.8 × 10 7 CFU/mL. In comparison to the qPCR method, the number of PCR cycles required for the constructed biosensor can be reduced to approximately 22 to achieve the same detection limit, effectively decreasing the detection time by about 24 min. The spiked recoveries in chicken were 91.8–106.0% with the relative standard deviations less than 10%, indicating that the constructed method exhibits good applicability. Graphical abstract
研究领域:
分子生物学
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生物传感技术
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微生物学
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DOI
PubMed
高影响因子
Food Chemistry: Molecular Sciences
·
2023
·
IF 3.3
Hanling Wang,Xianzhuo Meng,Li Yao,Qian Wu,Bangben Yao,Zhaoran Chen,Jianguo Xu,Wei Chen
Meat adulteration-based food fraud has recently become one of the global major economical, illegal, religious, and public health concerns. In this work, we developed a microarray chip polymerase chain reaction (PCR)-directed microfluidic lateral flow strip (LFS) device that facilitates the accurate and simultaneous identification of beef adulterated with chicken, duck, and pork, especially in processed beef products. To realize this goal, four pairs of amplification primers were designed and applied for specifically amplifying genomic DNA extracted from mixed meat powders in microarray chip. With the prominent advantage of this device lies in the flexible combination and integration of sample loading, detection, and reporting in microstructures, all the DNA amplicons can be individually visualized on the LFS unit, leading to the appearance of test lines (T C line, T D line, T P line, or T B line) as well as the control line (C line) for the species identification and quantification in beef products. Based on this new method, the adulterants were successfully distinguished and identified in mixtures down to 0.01% (wt.%) while the carryover aerogel contamination in routine molecular diagnostic laboratories was effectively avoided. The practicability, accuracy, and reliability of the device were further confirmed by using real-time PCR as a gold standard control on the successful identification of 50 processed ground meat samples sourced from local markets. The method and device proposed herein could be a useful tool for on-site identification of food authentication.
研究领域:
食品科学
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分子生物学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Zhenqing Li,Yifei Wang,Zehang Gao,Shinichi Sekine,Qingxiang You,Songlin Zhuang,Dawei Zhang,Shilun Feng,Yoshinori Yamaguchi
Background Rapid diagnosis of harmful microorganisms demonstrated its great importance for social health. Continuous flow PCR (CF-PCR) can realize rapid amplification of target genes by placing the microfluidic chip on heaters with different temperature. However, bubbles and evaporation always arise from heating, which makes the amplification not stable. Water-in-oil droplets running in CF-PCR microfluidic chip with uniform height takes long time because of the high resistance induced by long meandering microchannel . To overcome those drawbacks, we proposed a double-layer droplet CF-PCR microfluidic chip to reduce the fluidic resistance, and meanwhile nanoliter droplets were generated to minimize the bubbles and evaporation. Results Experiments showed that (1) fluidic resistance could be reduced with the increase of the height of the serpentine microchannel if the height of the T-junction part was certain. (2) Running speed, the size and the number of generated droplets were positively correlated with the cross-sectional area of the T-junction and water pressure. (3) Droplet fusion happened at higher water pressure if other experimental conditions were the same. (4) 0.032 nL droplet was created if the cross-sectional area of T-junction and water pressure were 1600 μm 2 (40 × 40 μm) and 7 kPa, respectively. Finally, we successfully amplified the target genes of Porphyromonas gingivalis within 11′16″ and observed the fluorescence from droplets. Significance and novelty Such a microfluidic chip can effectively reduce the high resistance induced by long meandering microchannel, and greatly save time required for droplets CF-PCR. It offers a new way for the rapid detection of bacterial.
研究领域:
生物医学工程
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分子生物学
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微流控技术
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诊断技术
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PubMed
高影响因子
BIOMEDICAL MICRODEVICES
·
2024
·Vol. 26
·期 2
·1-9
·
IF 3
Li Zhenqing,Ma Xiaolu,Zhang Zhen,Wang Xiaoyang,Yang Bo,Yang Jing,Zeng Yuan,Yuan Xujun,Zhang Dawei,Yamaguchi Yoshinori
Polymerase chain reaction (PCR) has been considered as the gold standard for detecting nucleic acids. The simple PCR system is of great significance for medical applications in remote areas, especially for the developing countries. Herein, we proposed a low-cost self-assembled platform for microchamber PCR. The working principle is rotating the chamber PCR microfluidic chip between two heaters with fixed temperature to solve the problem of low temperature variation rate. The system consists of two temperature controllers, a screw slide rail, a chamber array microfluidic chip and a self-built software. Such a system can be constructed at a cost of about US$60. The micro chamber PCR can be finished by rotating the microfluidic chip between two heaters with fixed temperature. Results demonstrated that the sensitivity of the temperature controller is 0.1℃. The relative error of the duration for the microfluidic chip was 0.02 s. Finally, we successfully finished amplification of the target gene of Porphyromonas gingivalis in the chamber PCR microfluidic chip within 35 min and on-site detection of its PCR products by fluorescence. The chip consisted of 3200 cylindrical chambers. The volume of reagent in each volume is as low as 0.628 nL. This work provides an effective method to reduce the amplification time required for micro chamber PCR.
研究领域:
生物医学工程
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分子生物学
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微流控技术
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医疗诊断
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DOI
PubMed
高影响因子
TALANTA
·
2020
·
IF 5.339
Qidi He,Meng Chen,Xiangan Lin,Zuanguang Chen
Single nucleotide polymorphism (SNP) analysis based on allele-specific polymerase chain reaction (AS-PCR) is a relatively effective and economical method compared with other genotyping technologies such as DNA sequencing, DNA hybridization and isothermal amplification strategies. But AS-PCR is limited by its labor-intensive optimization of reaction parameters and time-consuming result assessment. In this study, we put forward a novel idea of data processing to address this problem. SNP analysis was accomplished by AS-PCR with endpoint electrochemical detection . For each sample, two separate reactions were run simultaneously with two sets of allele-specific primers (wild-type primers for W system and mutant primers for M system). We measured their redox current signals on screen-printed electrodes once AS-PCR finished and calculated the difference value of current signals between two systems to determine the genotyping result. Based on the difference value of fluorescent signals, real-time fluorescent PCR was used to study reaction parameters in AS-PCR. With screened parameters, we obtained the genotyping results within 50 min. 36 hair-root samples from volunteers were analyzed by our method and their genotypes of ALDH2 gene (encoding aldehyde dehydrogenase 2) were totally identical with data from commercialized sequencing. Our work first employed difference value between two reaction systems to differentiate allele and provided a novel idea of data processing in AS-PCR method. It is able to promote the quick analysis of SNP in the fields of health monitor, disease precaution, and personalized diagnosis and treatment.
研究领域:
分子生物学
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遗传学
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生物医学工程
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Chuan Zhang,Kaixin Zheng,Chi Li,Ranran Zhang,Yicheng Zhu,Linxiao Xia,Yicheng Ma,Hans M. Wyss,Xiaoyu Cheng,Sailing He
Detecting proteins in ultralow concentrations in complex media is important for many applications but often relies on complicated techniques. Herein, a single-molecule protein analyzer with the potential for high-throughput applications is reported. Gold-coated magnetic nanoparticles with DNA-labeled antibodies were used for target recognition and separation. The immunocomplex was loaded into microdroplets generated with centrifugation. Immuno-PCR amplification of the DNA enabled the quantification of proteins at the level of single molecules. As an example, ultrasensitive detection of α-synuclein, a biomarker for neurodegenerative diseases, is achieved. The limit of detection was determined to be ∼50 aM in buffer and ∼170 aM in serum. The method exhibited high specificity and could be used to analyze post-translational modifications such as protein phosphorylation. This study will inspire wider studies on single-molecule protein detection, especially in disease diagnostics, biomarker discovery, and drug development.
研究领域:
生物技术
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分析化学
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诊断学
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生物标志物发现
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药物开发
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2021
·
IF 4.821
Zhengzheng Wang,Guoyang Xie,Guanhua Chen,Xingcai Gao,Jiaxin Li,Zongliang Xie,Hengyi Xu
Due to the massive use of antibiotics, clinical highly pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA) have emerged. Given its non-uniform drug resistance, it brings numerous difficulties for clinical diagnostic tests. This study aimed to establish a fast and accurate method to detect MRSA in hospital samples. In order to shorten the detection time and eliminate the influence of matrix on the detection results, so as to improve the detection sensitivity. In this study, magnetic separation (MS) technology was used for pre-treatment of samples, and triplex PCR was established using mecA , nuc and flic genes to realize the detection of MRSA. The results showed that the lowest detection limit of MRSA in blood samples and cerebrospinal fluid could reach 10 2 CFU/mL and 10 3 CFU/mL. Therefore, the MS technology combined with triplex PCR assay established in this study is expected to provide a strong basis for the detection of MRSA in hospital samples.
研究领域:
医学检验
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微生物学
·
临床诊断
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DOI
高影响因子
Science Advances
·
2024
·Vol. 10
·期 13
·
IF 11.7
Shun Hu,Liding Zhang,Ying Su,Xiaohan Liang,Jie Yang,Qingming Luo,Haiming Luo
Blood exosomes are emerging as potential biomarkers for diagnosing brain diseases such as Alzheimer’s disease (AD). There is currently a lack of an ultrasensitive technology for identifying core AD biomarkers in blood exosomes to optimize the utility of biomarkers in clinical practice. Here, an immunomagnetic exosomal polymerase chain reaction (iMEP) platform was developed using DNA-conjugated antibodies for the rapid detection of amyloid-β (Aβ1–40 and Aβ1–42) and phosphorylated tau (p-tau396,404 and p-tau181) in clinical blood exosomes. The toehold shift–mediated DNA affinity pulldown eliminates the high detection background, which allows the detection of biomarkers at concentrations down to 10 femtograms per milliliter. With the iMEP assay, exosomal Aβ1–42 was more accurate in differentiating patients with AD from healthy individuals compared with exosomal p-tau181 and p-tau396,404, with a sensitivity of 95.0% and a specificity of 95.0%. The iMEP technique is also adept at quantifying the levels of different exosomal biomarkers associated with disease pathogenesis.
研究领域:
神经科学
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分子生物学
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临床诊断
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生物标志物研究
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DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2022
·
IF 16.744
Xinglu Jiang,Kangjing Li,Beibei Xie,Jun Zhou
Taq DNA polymerase is the most widely used enzyme for PCR amplification and it contains 832 amino acids but no cysteines. Bovine serum albumin (BSA) is an 832-amino acid protein containing 35 cysteines, and at high temperature BSA can produce protein hydrogels through recombined disulfides and noncovalent bonds. When hydrogels are prepared using a mixture of Taq DNA polymerase and BSA, no disulfide bonds occur between them, and noncovalent bonds are the main forces holding together the Taq DNA polymerase with the hydrogels. During PCR amplification, temperature cycling can destroy the noncovalent bonds, thereby releasing Taq DNA polymerase from the hydrogels to synthesize DNA. The release rate, regulated through the variation of BSA concentration, allows for velocity-controlled amplification of DNA. As the amount of DNA products exceeds that of the Taq DNA polymerase released from hydrogels, a linear amplification replaces the exponential PCR amplification. When the amplification velocity begins to decrease, the time needed for reaching a plateau increase. This provides a broader detection window for the analysis of the end products, which correspond to the difference compared with the initial DNA concentration. The number of primers is continuously reduced with increasing cycles, which further causes a decline in the DNA amplification efficiency. The difference in the initial DNA template may cause a different consumption of primers, which can cause errors in the Ct values in the case of qPCR. Based on the slopes of the linear amplification curve, the Ct values can be calibrated to reduce this error, and a more accurate detection of the initial content of the target DNA can be implemented.
研究领域:
分子生物学
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生物技术
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生物化学
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DOI
高影响因子
危险品化学品经营许可证（带存储）