Rapid And high throughput RT-qPCR assay for identification and differentiation between SARS-CoV-2 variants B.1.1.7 and B.1.351

Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health activities and provide valuable data for epidemiological and policy decision making. We developed a multiplex quantitative RT-qPCR (qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions: one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, and one that detects the 242-244 deletion, which is specific to variant B.1.351. The fourth reaction identifies human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants, and not with other major known variants. The assay specificity was tested against a panel of respiratory pathogens (n=16), showing high specificity towards SC-2 RNA. The assay sensitivity was assessed using both In-vitro transcribed RNA and clinical samples, and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide circulating SC-2 variants.

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SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

Frontiers in Medicine ◽

10.3389/fmed.2021.627679 ◽

2021 ◽

Vol 8 ◽

Author(s):

Alfredo Garcia-Venzor ◽

Bertha Rueda-Zarazua ◽

Eduardo Marquez-Garcia ◽

Vilma Maldonado ◽

Angelica Moncada-Morales ◽

...

Keyword(s):

Limit Of Detection ◽

Direct Detection ◽

Direct Method ◽

Rna Extraction ◽

Rna Isolation ◽

High Sensitivity ◽

High Specificity ◽

Isothermal Amplification ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

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RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration

Viruses ◽

10.3390/v12080827 ◽

2020 ◽

Vol 12 (8) ◽

pp. 827

Author(s):

Paula Rodrigues de Almeida ◽

Ana Karolina Antunes Eisen ◽

Meriane Demoliner ◽

Fernando Rosado Spilki

Keyword(s):

Reverse Transcription ◽

Tissue Concentration ◽

Critical Role ◽

Rna Extraction ◽

High Sensitivity ◽

Clinical Samples ◽

Assay Sensitivity ◽

Robust Detection ◽

Detection Techniques ◽

Mosquito Pool

Zika virus (ZIKV) is an important arbovirus, responsible for recent outbreaks of Guillain Barré Syndrome and Congenital Zika Syndrome (CZS). After thousands of CZS cases, ZIKV is under constant surveillance in Brazil. Reliable and robust detection techniques are required to minimize the influence of host inhibitors from clinical samples and mosquito pool samples. Reverse transcription Digital Polymerase Chain Reaction (RT-dPCR) is a technique that allows the accurate quantification of DNA targets with high sensitivity, and it is usually less affected by inhibitors than RT-qPCR. This study aimed to assess the influence of mosquito tissue, RNA extraction and cDNA synthesis in ZIKV PCR detection. Samples containing 0, 3 and 10 mosquitoes were spiked with ZIKV MR766 and serially diluted prior to RNA extraction and RT-dPCR for ZIKV. Two reverse transcription protocols were tested. Assay sensitivity allowed the detection of 1.197 copies/µL. A higher correlation between dilution factor and target quantification was observed in 10 mosquito pool samples. The lower quantification in samples diluted without mosquitoes highlights the critical role of the reverse transcription step in RNA detection, since it could be attributed to reverse transcriptase variable performance in samples with low overall RNA concentration. The results in mosquito pools indicate that mosquito tissues do not inhibit ZIKV RT-dPCR, and the RT-dPCR technique has good sensitivity and robustness for ZIKV detection in mosquito pool samples regardless of mosquito tissue concentration.

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Post-antibiotic effect of marbofloxacin, enrofloxacin and amoxicillin against selected respiratory pathogens of pigs

Veterinární Medicína ◽

10.17221/123/2018-vetmed ◽

2019 ◽

Vol 64 (No. 02) ◽

pp. 67-77 ◽

Cited By ~ 1

Author(s):

K Nedbalcova ◽

M Zouharova ◽

D Sperling

Keyword(s):

Bacterial Infections ◽

Pasteurella Multocida ◽

Clinical Samples ◽

Respiratory Pathogens ◽

Minimal Inhibitory Concentrations ◽

Antibiotic Effect ◽

Significant Difference ◽

Effect Duration ◽

Post Antibiotic Effect

The post-antibiotic effect is defined as the period of bacterial growth suppression that persists after a limited exposure of organisms to antimicrobials and knowledge of its duration is important in establishing and optimising current dosing schedules for the treatment of bacterial infections. The post-antibiotic effect of marbofloxacin, enrofloxacin and amoxicillin were evaluated in vitro for Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida strains which originated from clinical samples of diseased pigs and were confirmed as susceptible to all tested antimicrobials based on determination of minimal inhibitory concentrations. The post-antibiotic effect for individual antimicrobials was monitored at five and ten times minimum inhibitory concentrations for one and two hours. The duration of the post-antibiotic effect for tested antimicrobials was found to exhibit the following order for all tested pathogens: marbofloxacin > enrofloxacin > amoxicillin. The longest duration of post-antibiotic effect of all tested antimicrobials was found in A. pleuropneumoniae and the shortest post-antibiotic effect duration was detected in P. multocida. No statistical differences in post-antibiotic effect duration were found within marbofloxacin and enrofloxacin in A. pleuropneumoniae and H. parasuis strains. In P. multocida strains there was a statistically significant difference (P = 0.0189). On the other hand, the differences between amoxicillin and marbofloxacin or enrofloxacin were statistically significant in all cases (P-values ranged between 0.0058 and 0.008). The prolonged post-antibiotic effect of fluoroquinolones and amoxicillin on important Gram-negative swine pathogens was confirmed. The results can be used to clarify the effect and mechanism of action of antimicrobial drugs in veterinary medicine.

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Rapid isothermal amplification and portable detection system for SARS-CoV-2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014739117 ◽

2020 ◽

Vol 117 (37) ◽

pp. 22727-22735 ◽

Cited By ~ 11

Author(s):

Anurup Ganguli ◽

Ariana Mostafa ◽

Jacob Berger ◽

Mehmet Y. Aydin ◽

Fu Sun ◽

...

Keyword(s):

Limit Of Detection ◽

Detection System ◽

Alternative Pathway ◽

Rna Extraction ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Complete Agreement ◽

Rt Pcr ◽

Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

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Utility of the Rapid Antigen Detection Test E. histolytica Quik Chek for the Diagnosis of Entamoeba histolytica Infection in Nonendemic Situations

Journal of Clinical Microbiology ◽

10.1128/jcm.01991-20 ◽

2020 ◽

Vol 58 (11) ◽

Author(s):

Yasuaki Yanagawa ◽

Rieko Shimogawara ◽

Tomoyuki Endo ◽

Rika Fukushima ◽

Hiroyuki Gatanaga ◽

...

Keyword(s):

Entamoeba Histolytica ◽

High Specificity ◽

Antigen Detection ◽

Transmitted Infection ◽

Cross Sectional ◽

Assay Sensitivity ◽

Content Type ◽

Rapid Antigen Detection Test ◽

Combined Use

ABSTRACT Entamoeba histolytica infection is an increasingly common sexually transmitted infection in Japan. Currently, stool ova and parasite examination (O&P) is the only approved diagnostic method. Here, we assessed the utility of the commercially available rapid antigen detection test (Quik Chek) for E. histolytica. A multicenter cross-sectional study was conducted. Stool samples that had been submitted for O&P were included. The samples were subjected to both Quik Chek and PCR, and the Quik Chek results were assessed in comparison with PCR as the reference standard. E. histolytica infection was confirmed in 5.8% (38/657) of the samples and comprised 20 diarrheal and 18 nondiarrheal cases. The overall sensitivity and specificity of Quik Chek were 44.7% (95% confidence interval, 30.1 to 60.3) and 99.8% (99.1 to 100), respectively. The sensitivity of Quik Chek was higher for diarrheal cases (60.0%) than for nondiarrheal cases (27.8%). Furthermore, the combined use of Quik Chek with O&P increased the sensitivity (78.9%), especially for diarrheal cases (up to 90%). The E. histolytica burden assessed by quantitative PCR was similar between Quik Chek-positive and -negative samples. The Quik Chek assay sensitivity was lower for cyst-containing stools than for trophozoite-containing stools, although it was shown that cultured E. histolytica clinical strains from Quik Chek-negative cyst-containing stools exhibited antigenicity in vitro. The present study confirmed the high specificity of Quik Chek for E. histolytica infection. Combined use with O&P increased the sensitivity of detection, facilitating the use of Quik Chek in point-of-care settings in nonendemic situations.

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Evaluation of a DNA Microarray, the Check-Points ESBL/KPC Array, for Rapid Detection of TEM, SHV, and CTX-M Extended-Spectrum β-Lactamases and KPC Carbapenemases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01298-09 ◽

2010 ◽

Vol 54 (8) ◽

pp. 3086-3092 ◽

Cited By ~ 65

Author(s):

T. Naas ◽

G. Cuzon ◽

H. Truong ◽

S. Bernabeu ◽

P. Nordmann

Keyword(s):

High Specificity ◽

Rapid Identification ◽

Clinical Samples ◽

Genetic Profiling ◽

Fast Detection ◽

Extended Spectrum ◽

Check Points ◽

Total Dna ◽

Working Day ◽

Esbl Producers

ABSTRACT Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ß-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ß-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ß-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.

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Diagnostic Performance and Clinical Feasibility of a Novel One-Step RT-qPCR for Detecting Multiple Severe Acute Respiratory Syndrome Coronavirus

10.21203/rs.3.rs-1027593/v1 ◽

2021 ◽

Author(s):

Tran Bac Le ◽

Hye Kwon Kim ◽

Min-Ju Ahn ◽

Mark Zanin ◽

Van Thi Lo ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Limit Of Detection ◽

Laboratory Diagnosis ◽

Rapid Identification ◽

Qpcr Assay ◽

Clinical Samples ◽

Reverse Transcription Pcr ◽

One Step ◽

Human Coronaviruses ◽

Culture Supernatants

Abstract The pandemic coronavirus disease 2019 (COVID-19) is characterized as an acute respiratory infection in the majority of cases and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other coronaviruses (CoVs) can infect humans, although the majority only cause mild respiratory symptoms. As early diagnosis of SARS-CoV-2 is critical to prevent further transmission events and to improve clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other CoVs in respiratory samples. Therefore, we developed and evaluated a novel quantitative reverse transcription PCR (RT-qPCR) assay, targeting the spike (S) and membrane (M) genes, to enable the rapid identification of SARS-CoV-2 including several new circulating variants, and other emerging pan-SARS-like CoVs. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays confirmed a limit of detection of 1 × 100 TCID50/ml and no cross-reaction with human coronaviruses or other respiratory viruses. We also validated our method using human clinical samples from COVID-19 patients and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic coronaviruses, including SARS-CoV-2, and may be useful for the identification of other pan-SARS-like CoVs of zoonotic origin.

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The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650659 ◽

1996 ◽

Vol 76 (05) ◽

pp. 774-779 ◽

Cited By ~ 14

Author(s):

John T Brandt ◽

Carmen J Julius ◽

Jeanne M Osborne ◽

Clark L Anderson

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Complement Activation ◽

Thromboembolic Disease ◽

Clinical Samples ◽

Hla Antigen ◽

Aggregation Response ◽

Immune Mediated ◽

Dependent Pathway

SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

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A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650145 ◽

1981 ◽

Vol 45 (02) ◽

pp. 110-115 ◽

Cited By ~ 5

Author(s):

György Csákó ◽

Eva A Suba

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Platelet Reactivity ◽

High Specificity ◽

Turbidimetric Method ◽

Specific Manner ◽

Aggregation Inhibition ◽

Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

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Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects

Protein and Peptide Letters ◽

10.2174/0929866527666191223141205 ◽

2020 ◽

Vol 27 (5) ◽

pp. 400-410

Author(s):

Valentina De Luca ◽

Luigi Mandrich

Keyword(s):

Gene Evolution ◽

Sequence Divergence ◽

High Specificity ◽

Industrial Applications ◽

Point Of View ◽

Enzyme Promiscuity ◽

Primary Activity ◽

Starting Point ◽

Main Activity

: Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

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