Detection and quantification of EGFR T790M mutation

Detection and quantification of EGFR T790M mutation in liquid biopsies by droplet digital PCR

 Background: Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with advanced non-small cell lung cancer (NSCLC), droplet digital PCR (ddPCR) is increasingly used to genotype the epidermal growth factor receptor (EGFR) gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. The aim of this study was to implement and assess the performance of a ddPCR assay for detecting the EGFR T790M mutation in liquid biopsies.

 Methods: A ddPCR assay was optimized to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression.

 Results: Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%.

The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases.

Conclusions: This study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis.

A rapid and sensitive fluorescence biosensor based on plasmonic PCR

  •  Plasmonic PCR utilizing metallic nanoparticles has shown great advantages compared to the commercial thermocycler equipment in terms of cost, size and processing time. However, due to the strong fluorescence quenching, plasmonic nanoparticle-based PCR requires additional post-processing steps such as centrifugation and gel electrophoresis.
  • This process increases the overall diagnostic time, offsetting the benefits of fast thermocycling. Here, we report a rapid and sensitive plasmonic photothermal PCR (PPT-PCR) assay method based on in situ end-point fluorescence detection.
  • By using plasmonic magnetic bi-functional nanoparticles, PPT-PCR involving 30 thermocycles and fluorescence detection following magnetic separation has successfully shown that DNA targets can be detected within 5.5 minutes.
  • The limit of detection (3.3 copies per μL) is comparable with that of the conventional real-time quantitative PCR; however, the assay time is about 5.5 times shorter for the PPT-PCR. The strategy of combining the photothermal effect and magnetic separation into a single particle will open new horizons in the development of fast and sensitive PCR-based biosensors for point-of care testing.
myology2019
myology2019

Comorbidities and Age Are Associated With Persistent COVID-19 PCR Positivity

 Objectives: The impact of demographics and comorbidities on the duration of COVID-19 nasopharyngeal swab PCR positivity remains unclear. The objective of our analysis is to determine the impact of age, intensive care unit (ICU) admission, comorbidities, and ethnicity on the duration of COVID-19 PCR positivity among hospitalized patients in a large group of hospital.

 Method: We studied 530 patients from a large hospital system and time to SARS-CoV-2 virus RNA PCR negativity at any-time during hospitalization or following discharge from the hospital was the primary endpoint. We included patients 18 years or older who tested positive for COVID-19 during an inpatient, outpatient, or emergency room visit between February 1, 2020, and April 14, 2020.

 Results: Overall, 315 (59.4%) of our patient population continued to have a positive SARS-CoV-2 virus RNA PCR 4 weeks after the initial positive test. We found that age>70 years, chronic kidney disease, hypertension, hyperlipidemia, obesity, or coronary artery disease are associated with persistent PCR positivity for more than 4 weeks after initial diagnosis.

 Conclusion: Age, and the presence of co-morbidities should be taken into consideration when interpreting a positive COVID PCR test.

Comparison of PCR and phenotypic methods for the detection of methicillin resistant Staphylococcus aureus

 Background and objectives: Resistance to methicillin in methicillin resistant strains of Staphylococcus aureus (MRSA) is due to the presence of mec-A gene, which encodes a low affinity penicillin binding protein (PBP)-2a or PBP2. Accurate and rapid identification of MRSA in clinical specimens is essential for timely decision on effective treatment. The aim of the study was to compare three different methods for detection of MRSA namely cefoxitin disc diffusion, CHROM agar MRSA and VITEK-2 susceptibility with PCR which is the gold standard reference method and to find the antibiotic susceptibility pattern of these isolates by VITEK-2.

 

Materials and methods: A Total of 100 non-duplicate S. aureus isolates were collected from different clinical samples among both outpatient and inpatients. Detection of MRSA among these isolates was done by cefoxitin disc diffusion, VITEK-2, CHROM agar MRSA and PCR.

 Results: The sensitivity and specificity of cefoxitin disc diffusion and Vitek was found to be 97.2% and 100%, while that of CHROM agar was found to be 100% and 78.6%. The overall prevalence of MRSA in our study by PCR was 72%.

 

Conclusion: Based on the findings in our study, isolates which show cefoxitin zone diameter < 22 mm can be reported as MRSA. However, those isolates which have a zone diameter between 22-24 mm, should ideally be confirmed by PCR.

iFluor™ 546 succinimidyl ester

1048 1 mg
EUR 219

iFluor™ 568 succinimidyl ester

1049 1 mg
EUR 219

iFluor™ 450 maleimide

1057 1 mg
EUR 219

iFluor™ 647 amine

1074 1 mg
EUR 219

iFluor™ 680 amine

1076 1 mg
EUR 219

iFluor™ 700 amine

1077 1 mg
EUR 219

iFluor™ 710 amine

1078 1 mg
EUR 219

iFluor™ 750 amine

1079 1 mg
EUR 219

iFluor™ 680 hydrazide

1086 1 mg
EUR 219

iFluor™ 700 hydrazide

1087 1 mg
EUR 219

iFluor™ 750 hydrazide

1088 1 mg
EUR 219

ReadiUse™ ABTS Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*

11001 1 L
EUR 202

ReadiUse™ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*

11003 1 L
EUR 480

ReadiUse™ hydrogen peroxide solution *50 mM calibrated and stabilized solution*

11004 5x10 mL
EUR 132

Amplite™ Blue

11005 25 mg
EUR 176

Amplite™ IR

11009 1 mg
EUR 132

Amplite™ Red

11011 1000 Assays
EUR 132

ReadiUse™ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*

11012 100 ml
EUR 115

ReadiLink™ Rapid mFluor™ Violet 420 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1105 2 Labelings
EUR 176

Luminol [3-Aminophthalhydrazide] *CAS 521-31-3*

11050 1 g
EUR 115

iFluor™ 488 tyramide

11060 1 mg
EUR 219

Azido-Cy5 tyramide

11061 1 mg
EUR 306

Amplite™ Fluorimetric Fluorescamine Protein Quantitation Kit *Blue Fluorescence*

11100 200 Tests
EUR 219

Amplite™ Fluorimetric Protein Quantitation Kit *Orange Fluorescence*

11105 500 tests
EUR 219

ReadiLink™ Rapid mFluor™ Violet 540 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1114 2 Labelings
EUR 176

ReadiLink™ Rapid mFluor™ Blue 570 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1120 2 Labelings
EUR 176

ReadiLink™ Rapid mFluor™ Green 620 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1123 2 Labelings
EUR 176

ReadiLink™ Rapid mFluor™ Yellow 630 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1126 2 Labelings
EUR 50

Amplite™ Colorimetric Glucose Oxidase Assay Kit

11299 500 Tests
EUR 306

ReadiLink™ Rapid mFluor™ Red 700 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1130 2 Labelings
EUR 176

Amplite™ Fluorimetric Glucose Oxidase Assay Kit *Red Fluorescence*

11300 500 Tests
EUR 219

Amplite™ Fluorimetric Myeloperoxidase Assay Kit *Red Fluorescence*

11301 200 Tests
EUR 219

Amplite™ Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*

11302 200 Tests
EUR 219

Amplite™ Fluorimetric Monoamine Oxidase Assay Kit *Red Fluorescence*

11303 200 Tests
EUR 219

Amplite™ Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*

11304 200 Tests
EUR 219

Amplite™ Colorimetric Superoxide Dismutase (SOD) Assay Kit

11305 200 Tests
EUR 219

Amplite™ Fluorimetric Catalase Assay Kit *Red Fluorescence*

11306 200 Tests
EUR 219

Amplite™ Colorimetric Xanthine Oxidase Assay Kit

11307 200 Tests
EUR 219

mFluor™ Violet 450 acid

1140 5 mg
EUR 219

Amplite™ Colorimetric Acetylcholinesterase Assay Kit

11400 200 Tests
EUR 219

Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Green Fluorescence*

11401 200 Tests
EUR 219

Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*

11402 200 Tests
EUR 219

Amplite™ Fluorimetric Acetylcholine Assay Kit *Red Fluorescence*

11403 200 Tests
EUR 219

mFluor™ Violet 510 acid

1141 5 mg
EUR 306

mFluor™ Violet 540 acid

1142 5 mg
EUR 306

mFluor™ Blue 570 acid

1143 5 mg
EUR 306

mFluor™ Green 620 acid

1144 5 mg
EUR 306

mFluor™ Yellow 630 acid

1145 5 mg
EUR 50

mFluor™ Red 700 acid

1146 5 mg
EUR 306

mFluor™ Violet 500 SE

1149 1 mg
EUR 219

mFluor™ Violet 450 SE

1150 1 mg
EUR 219

Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*

11503 200 Tests
EUR 263

Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence*

11504 100 Tests
EUR 263

Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence Optimized for Flow Cytometry*

11505 100 Tests
EUR 263

Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence Optimized for Flow Cytometry*

11506 100 Tests
EUR 263

mFluor™ Violet 510 SE

1151 1 mg
EUR 219

mFluor™ Violet 540 SE

1152 1 mg
EUR 219

Amplite™ Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*

11540 1000 Tests
EUR 219

Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*

11541 1000 Tests
EUR 219

Amplite™ Colorimetric Peroxidase (HRP) Assay Kit *Blue Color*

11551 500 Tests
EUR 219

Amplite™ Fluorimetric Peroxidase (HRP) Assay Kit *Red Fluorescence*

11552 500 Tests
EUR 219

Amplite™ Fluorimetric Peroxidase (HRP) Assay Kit *Near Infrared Fluorescence*

11553 500 Tests
EUR 306

Amplite™ Luminometric Peroxidase (HRP) Assay Kit

11559 500 Tests
EUR 219

Amplite™ Fluorimetric Glutathione Peroxidase Assay Kit *Blue Fluorescence*

11560 200 Tests
EUR 306

mFluor™ Blue 570 SE

1160 1 mg
EUR 306

FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*

11600 5 mg
EUR 132

MUP, disodium salt [4-Methylumbelliferyl phosphate, disodium salt] *CAS 22919-26-2*

11610 25 mg
EUR 115

MUP, disodium salt [4-Methylumbelliferyl phosphate, disodium salt] *CAS 22919-26-2*

11612 10 g
EUR 898

MUP [4-Methylumbelliferyl phosphate, free acid] *CAS 3368-04-5*

11614 25 mg
EUR 115

MUP [4-Methylumbelliferyl phosphate, free acid] *CAS 3368-04-5*

11617 5 g
EUR 480

pNPP [4-Nitrophenyl phosphate, disodium salt] *CAS 4264-83-9*

11619 25 mg
EUR 115

CF-MUP, sodium salt *Superior alternative to MUP*

11628 10 mg
EUR 50

mFluor™ Green 620 SE

1165 1 mg
EUR 306

mFluor™ Yellow 630 SE

1170 1 mg
EUR 50

ADP-ribose-pNP

11700 1 mg
EUR 219

mFluor™ Red 780 SE

1191 1 mg
EUR 306

Amplite™ Colorimetric Alkaline Phosphatase Assay Kit *Yellow Color*

11950 500 Tests
EUR 176

Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Blue Fluorescence*

11952 500 Tests
EUR 219

Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*

11953 500 Tests
EUR 219

Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Near Infrared Fluorescence*

11954 500 Tests
EUR 219

Amplite™ Luminometric Alkaline Phosphatase Assay Kit *Luminescence*

11956 100 Tests
EUR 219

ReadiLink™ Rapid iFluor™ 555 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1227 2 Labelings
EUR 176

ReadiLink™ Rapid iFluor™ 647 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1235 2 Labelings
EUR 176

ReadiLink™ Rapid iFluor™ 680 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1240 2 Labelings
EUR 176

ReadiLink™ Rapid iFluor™ 700 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1245 2 Labelings
EUR 176

Cyluc1 [(4S)-2-(6,7-Dihydro-5H-thiazolo[4,5-f]indol-2-yl)-4,5-dihydro-thiazole-4-carboxylic acid]

12481 25 mg
EUR 219

Cyluc1 [(4S)-2-(6,7-Dihydro-5H-thiazolo[4,5-f]indol-2-yl)-4,5-dihydro-thiazole-4-carboxylic acid]

12482 100 mg
EUR 480

ReadiLink™ Rapid iFluor™ 750 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

1250 2 Labelings
EUR 176

D-Luciferin, free acid *CAS#: 2591-17-5*

12501 25 mg
EUR 115

D-Luciferin, free acid *CAS#: 2591-17-5*

12502 100 mg
EUR 132

Universal primers for rapid detection of six pospiviroids in Solanaceae plants using one-step RT-PCR and RT-LAMP

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are currently listed as quarantine pathogens in many countries. Among them, columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd) are of major concerns.

The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids.

The minimal concentration of viroid RNA required for a successful detection varied, ranging from one femtogram to 10 nanograms, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR but both assays were rapid and highly sensitive tools to detect six pospiviroids.

Detection methods currently in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate to screen a large number of solanaceous plants and seeds intended for import and export.

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