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.

EUGONIC AGAR

E05-106-2Kg 2 Kg Ask for price

EUGONIC AGAR

E05-106-500g 500 g Ask for price

ENDO AGAR

E05-110-10kg 10 kg
EUR 2381

ENDO AGAR

E05-110-2Kg 2 Kg
EUR 557

ENDO AGAR

E05-110-500g 500 g
EUR 188

APT AGAR

A01-101-10kg 10 kg
EUR 1173

APT AGAR

A01-101-2Kg 2 Kg
EUR 294

APT AGAR

A01-101-500g 500 g
EUR 116

AGAR, BACTERIOLOGICAL

A01-102-10kg 10 kg
EUR 1684

AGAR, BACTERIOLOGICAL

A01-102-2kg 2kg
EUR 405

AGAR, BACTERIOLOGICAL

A01-102-500g 500 g
EUR 146

AGAR, HIGH GEL

A01-102IHG-10kg 10 kg
EUR 2219

AGAR, HIGH GEL

A01-102IHG-2Kg 2 Kg
EUR 521

AGAR, HIGH GEL

A01-102IHG-500g 500 g
EUR 178

AGAR,NOBLE

A01-102N-10kg 10 kg
EUR 3910

AGAR,NOBLE

A01-102N-2kg 2kg
EUR 889

AGAR,NOBLE

A01-102N-500g 500 g
EUR 278

ALT. THIOGLYCOLLATE MED.

A01-103-10kg 10 kg Ask for price

ALT. THIOGLYCOLLATE MED.

A01-103-2Kg 2 Kg Ask for price

ALT. THIOGLYCOLLATE MED.

A01-103-500g 500 g Ask for price

AMIES BROTH WITH CHARCOAL

A01-104-10kg 10 kg Ask for price

AMIES BROTH WITH CHARCOAL

A01-104-2kg 2kg Ask for price

AMIES BROTH WITH CHARCOAL

A01-104-500g 500 g Ask for price

AMIES BROTH W/O CHARCOAL

A01-105-10kg 10 kg Ask for price

AMIES BROTH W/O CHARCOAL

A01-105-2Kg 2 Kg Ask for price

AMIES BROTH W/O CHARCOAL

A01-105-500g 500 g Ask for price

AZIDE BLOOD AGAR BASE

A01-113-10kg 10 kg
EUR 2532

AZIDE BLOOD AGAR BASE

A01-113-2kg 2kg
EUR 589

AZIDE BLOOD AGAR BASE

A01-113-500g 500 g
EUR 197

AZIDE DEXTROSE BROTH

A01-114-10kg 10 kg
EUR 868

AZIDE DEXTROSE BROTH

A01-114-2Kg 2 Kg
EUR 228

AZIDE DEXTROSE BROTH

A01-114-500g 500 g
EUR 98

ANAEROBIC AGAR

A01-115-10kg 10 kg
EUR 2556

ANAEROBIC AGAR

A01-115-2kg 2kg
EUR 595

ANAEROBIC AGAR

A01-115-500g 500 g
EUR 198

A-1 MEDIUM

A01-116-10kg 10 kg
EUR 1034

A-1 MEDIUM

A01-116-2Kg 2 Kg
EUR 264

A-1 MEDIUM

A01-116-500g 500 g
EUR 108

AGAROSE 25G-125G -500G

A01-118-10kg 10 kg Ask for price

AGAROSE 25G-125G -500G

A01-118-2kg 2kg Ask for price

AGAROSE 25G-125G -500G

A01-118-500g 500 g
EUR 494

IL-2 Receptor ?, Human Recombinant

7100-10
EUR 196

IL-2 Receptor ?, Human Recombinant

7100-50
EUR 637

Human CellExp? IL-4 R?, Human Recombinant

7101-10
EUR 229

Human CellExp? IL-4 R?, Human Recombinant

7101-50
EUR 881

Human CellExp? IL-6 R?, Human Recombinant

7102-10
EUR 207

Human CellExp? IL-6 R?, Human Recombinant

7102-50
EUR 800

IL-9, Rat Recombinant

7103-10
EUR 278

IL-9, Rat Recombinant

7103-50
EUR 1132

IL-12p80, Human Recombinant

7104-10
EUR 392

IL-12p80, Human Recombinant

7104-50
EUR 1675

IL-17A, Mouse Recombinant

7105-10
EUR 196

IL-17A, Mouse Recombinant

7105-50
EUR 675

IL-17B, Human Recombinant

7106-10
EUR 196

IL-17B, Human Recombinant

7106-50
EUR 675

IL-17D, Human Recombinant

7107-10
EUR 196

IL-17D, Human Recombinant

7107-50
EUR 675

Human CellExp? IL-34, Human Recombinant

7108-10
EUR 278

Human CellExp? IL-34, Human Recombinant

7108-50
EUR 1132

IL-36RA, Human Recombinant

7109-10
EUR 196

IL-36RA, Human Recombinant

7109-50
EUR 675

IL-36?, Human Recombinant

7110-10
EUR 278

IL-36?, Human Recombinant

7110-50
EUR 1132

IL-36?, Human Recombinant

7111-10
EUR 278

IL-36?, Human Recombinant

7111-50
EUR 1132

Activin B, Human Recombinant

7112-10
EUR 479

Activin B, Human Recombinant

7112-50
EUR 2094

AITRL, Human Recombinant

7113-10
EUR 251

AITRL, Human Recombinant

7113-50
EUR 936

Amphiregulin, Human Recombinant

7114-10
EUR 147

Amphiregulin, Human Recombinant

7114-50
EUR 376

ANG-1, Human Recombinant

7115-10
EUR 251

ANG-1, Human Recombinant

7115-50
EUR 936

ANG-2, Human Recombinant

7116-10
EUR 251

ANG-2, Human Recombinant

7116-50
EUR 936

APRIL, Human Recombinant

7117-10
EUR 311

APRIL, Human Recombinant

7117-50
EUR 1300

APRIL, Mouse Recombinant

7118-10
EUR 196

APRIL, Mouse Recombinant

7118-50
EUR 675

Betacellulin, Murine Recombinant

7119-10
EUR 196

Betacellulin, Murine Recombinant

7119-50
EUR 675

Cardiotrophin-1, Murine Recombinant

7121-10
EUR 278

Cardiotrophin-1, Murine Recombinant

7121-50
EUR 1132

sCD100/Semaphorin-4D, Human Recombinant

7126-10
EUR 251

sCD100/Semaphorin-4D, Human Recombinant

7126-50
EUR 936

CTGFL/WISP-2, Human Recombinant

7129-10
EUR 234

CTGFL/WISP-2, Human Recombinant

7129-50
EUR 860

Human CellExp? DKK-1, Human Recombinant

7132-10
EUR 278

Human CellExp? DKK-1, Human Recombinant

7132-50
EUR 1132

Human CellExp? sDLL-1, Human Recombinant

7133-10
EUR 207

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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