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1.
Front Cell Dev Biol ; 9: 659809, 2021.
Article in English | MEDLINE | ID: covidwho-1285273

ABSTRACT

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through interactions with its receptor, Angiotensin-converting enzyme 2 (ACE2), causing severe acute respiratory syndrome and death in a considerable proportion of people. Patients infected with SARS-CoV-2 experience digestive symptoms. However, the precise protein expression atlas of ACE2 in the gastrointestinal tract remains unclear. In this study, we aimed to explore the ACE2 protein expression pattern and the underlying function of ACE2 in the gastrointestinal tract, including the colon, stomach, liver, and pancreas. Methods: We measured the protein expression of ACE2 in the gastrointestinal tract using immunohistochemical (IHC) staining with an ACE2-specific antibody of paraffin-embedded colon, stomach, liver, and pancreatic tissues. The correlation between the protein expression of ACE2 and the prognosis of patients with gastrointestinal cancers was analyzed by the log-rank (Mantel-Cox) test. The influence of ACE2 on colon, stomach, liver, and pancreatic tumor cell line proliferation was tested using a Cell Counting Kit 8 (CCK-8) assay. Results: ACE2 presented heterogeneous expression patterns in the gastrointestinal tract, and it showed a punctate distribution in hepatic cells. Compared to that in parallel adjacent non-tumor tissues, the protein expression of ACE2 was significantly increased in colon cancer, stomach cancer, and pancreatic cancer tissues but dramatically decreased in liver cancer tissues. However, the expression level of the ACE2 protein was not correlated with the survival of patients with gastrointestinal cancers. Consistently, ACE2 did not affect the proliferation of gastrointestinal cancer cells in vitro. Conclusion: The ACE2 protein is widely expressed in the gastrointestinal tract, and its expression is significantly altered in gastrointestinal tumor tissues. ACE2 is not an independent prognostic marker of gastrointestinal cancers.

2.
Journal of Modern Laboratory Medicine ; 35(5):113-117, 2020.
Article in Chinese | GIM | ID: covidwho-1264598

ABSTRACT

Objective: To evaluate whether a domestic novel coronavirus (SARS-CoV-2) nucleic acid detection kit meets the requirements of clinical SARS-CoV-2 nucleic acid detection.

3.
China Tropical Medicine ; 21(3):299-302, 2021.
Article in Chinese | GIM | ID: covidwho-1236988

ABSTRACT

Objective: Clinical value of SARS-CoV-2 nucleic acid detection combined with specific IgM/IgG in the diagnosis of three asymptomatic COVID-19 infections was investigated.

4.
Bioact Mater ; 6(12): 4580-4590, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1230373

ABSTRACT

CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction. Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.

5.
Journal of Modern Laboratory Medicine ; 35(4):100-105, 2020.
Article in Chinese | GIM | ID: covidwho-1073553

ABSTRACT

Objective: To evaluate the performance of three kinds of chemiluminescence immunoassay (CLIA) kits for novel Coronavirus (SARS-COV-2) antibody, and study the clinical application of SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies detection.

6.
PLoS Biol ; 18(12): e3000978, 2020 12.
Article in English | MEDLINE | ID: covidwho-1044757

ABSTRACT

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.


Subject(s)
COVID-19 Testing/methods , CRISPR-Cas Systems/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Base Sequence , Humans , Reproducibility of Results , Sensitivity and Specificity
7.
J Clin Lab Anal ; 35(1): e23681, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-986204

ABSTRACT

BACKGROUND: Seldom performance evaluation and diagnosis comparison studies were reported for different chemiluminescent immunoassay (CLIA) kits approved under an emergency approval program for SARS-CoV-2 infection. METHODS: A total of 100 and 105 serum separately from non-infected populations and COVID-19 patients were detected with SARS-CoV-2 IgM and IgG kits. The characteristics including precision, functional sensitivity, linearity, and accuracy were evaluated for Axceed, iFlash, and Maglumi CLIA kits. RESULTS: Maglumi and iFlash had the best analytical sensitivity for IgM and IgG, respectively. Axceed kits had a linearity response in the detected concentration. The clinical sensitivity of Axceed, iFlash, and Maglumi was 68.0%, 64.9%, and 63.9% with a specificity of 99.0%, 96.0%, and 100% for IgM, 85.6%, 97.9%, and 94.8% with a specificity of 97.0% for IgG. ROC analysis indicated all kits had a diagnostic power greater than 0.9. Notably, either IgM or IgG kits obtained a poor agreement (Kappa value from 0.397 to 0.713) with others. Among 38 recovered patients, 94.7% had an effective immune response, and both seropositive IgM and IgG accounted for the biggest proportion (medium, 42 days onset), then followed by the single seropositive IgG (medium, 50 days onset) in Ab profile. CONCLUSION: The performance of CLIA kits satisfied the diagnosis of SARS-CoV-2 infection. Both positive of IgG and IgM contributes to improve the specificity, and a positive one will enhance the sensitivity.


Subject(s)
COVID-19 Testing/methods , COVID-19/etiology , Immunoassay/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , Adult , Aged , Antibodies, Viral/blood , Automation, Laboratory , COVID-19/diagnosis , Female , Humans , Luminescence , Pregnancy , Pregnancy Complications, Infectious/etiology , Pregnancy Complications, Infectious/therapy , Reproducibility of Results , SARS-CoV-2/immunology , Time Factors
8.
J Clin Lab Anal ; 35(1): e23643, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-891884

ABSTRACT

BACKGROUND: We aimed to evaluate the analytical performance of five commercial RT-PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. METHODS: A total of 20 COVID-19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT-PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross-reactivity. RESULTS: The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS-CoV-2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross-reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. CONCLUSIONS: Five RT-PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS-CoV-2.


Subject(s)
COVID-19 Testing/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Humans , Limit of Detection , Nasopharynx/virology , Polyproteins/genetics , Reproducibility of Results , Viral Proteins/genetics
9.
EBioMedicine ; 61: 103036, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-844322

ABSTRACT

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Subject(s)
Bacterial Proteins/metabolism , Betacoronavirus/genetics , CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endodeoxyribonucleases/metabolism , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Limit of Detection , Nasal Cavity/virology , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , Reference Standards , SARS-CoV-2 , Viral Proteins/genetics , Viral Proteins/metabolism
10.
Microb Biotechnol ; 13(4): 950-961, 2020 07.
Article in English | MEDLINE | ID: covidwho-116666

ABSTRACT

The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Betacoronavirus/classification , Betacoronavirus/genetics , COVID-19 , China , Coronavirus Infections/virology , DNA Primers/genetics , Humans , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and Specificity , Viral Proteins/genetics
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