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.

G. Pig IgG Fc-Biotin conjugate (isotype control, non-immune) purified
20004-3-B 0.1 mg
EUR 225
G. Pig IgG-Biotin conjugate (isotype control)
20004-B 100 ug
EUR 202
Rat IgG F(ab')2 fragment, purified (isotype control)
20005-1-FAB2 1 mg
EUR 202
Rat IgG (Fc)-Biotin Conjuagte (isotype control), purified
20005-1-FC-B 0.1 mg
EUR 225
Rat IgG1-Biotin conjugate (isotype control)
20005-11-B 100 ug
EUR 202
Rat IgG2a-APC conjugate (isotype control)
20005-12-APC 25 tests
EUR 202
Rat IgG2a-Biotin conjugate (isotype control)
20005-12-B 100 ug
EUR 202
Rat IgG2b-Biotin conjugate (isotype control)
20005-13-B 100 ug
EUR 202
Rat IgG2c-Biotin conjugate (isotype control)
20005-14-B 100 ug
EUR 225
Rat IgM-Biotin conjugate (isotype control)
20005-21-B 100 ug
EUR 225
Rat IgG-Agarose conjugate(aff matrix)
20005-AS-1 0.5 ml
EUR 164
Rat IgG-Biotin conjugate (isotype control) (Isotype control)
20005-B 100 ug
EUR 164
Sheep IgG-Biotin Conjugate (isotype control, non-immune), purified
20006-1-B 0.5 mg
EUR 202
Sheep IgM-Biotin Conjugate (non-immune) control, purified
20006-2-B 0.1 mg
EUR 225
Sheep IgG Fc-Biotin Conjugate (isotype control, non-immune), purified
20006-4-B 0.5 mg
EUR 202
Human IgG-Biotin conjugate (isotype control, non-immune), purified
20007-1-B 0.5 mg
EUR 225
Human IgG (>98%, non-immune, control, Liquid @ 10 mg/ml, azide free, bulk size)
20007-1-BL-1 10 ml Ask for price
Human IgG (>98%, non-immune, control, powder, azide free, bulk size)
20007-1-BP-1 1 g
EUR 651
Human IgG (>98%, non-immune, control, powder, azide free, bulk size)
20007-1-BP-10 10 g
EUR 4313
Human IgG Fab fragment-Biotin Conjugate, purified
20007-1-FAB-B 0.1 mg
EUR 202
Human IgG F(ab')2 fragment, purified
20007-1-FAB2 1 mg
EUR 202
Human IgG Fab2 fragment-Biotin Conjugate, purified
20007-1-FAB2-B 0.1 mg
EUR 202
Human IgG Fc-Biotin conjugate (isotype control, non-immune), purified
20007-1-Fc-B 0.1 mg
EUR 164
Human IgG (Fc) fragment, purified (>95%, low endotoxin)
20007-1-FC-LE 100 ul
EUR 225
Human IgG (Fc) fragment-Biotin purified (>95%, low endotoxin)
20007-1-FC-LE-B 25 ug
EUR 286
Human IgG (serum origin, purified >97%, low endotoxin, azide free)
20007-1-LE-1 1 mg
EUR 164
Human IgG-Biotin (serum origin, purified >97%, low endotoxin, azide free)
20007-1-LE-BTN 100 ug
EUR 286
TruStrip RDT Human IgG Rapid test cards, 10/pk
20007-1-RDT 1 Pk
EUR 171
TruStrip RDT Human IgG Rapid test cards, 25/pk
20007-1-RDT-25 1 Pk
EUR 293
Human IgM, purified (native, isotype control, >96% pure, ELISA Grade)
20007-2 1 mg
EUR 164
Human IgM purified (isotype control, >96% pure, ELISA Grade)
20007-2-10 10 mg
EUR 895
Human IgM purified (isotype control, >96% pure, ELISA Grade)
20007-2-5 5 mg
EUR 529
Human IgM (myeloma, >96%, non-immune, control; bulk size)
20007-2-B-100 100 mg Ask for price
Human IgM (myeloma, >96%, non-immune, control; bulk size)
20007-2-B-1000 1000 mg Ask for price
Human IgM-Biotin conjugate (myeloma, isotype control)
20007-2-MB 100 ug
EUR 164
Human IgM (Rheumatoid Factor plasma, >40,000 IU/ml, purifed for ELISA)
20007-2-RF 100 ul
EUR 469
Human IgE (myeloma, kappa), purified (isotype control, >95%, low endotoxin, azide free)
20007-3-LE-50 100 ug
EUR 408
Human IgE-Biotin (myeloma, kappa), purified (isotype control, >95%, low endotoxin, azide free)
20007-3-LE-BTN 100 ul
EUR 408
Human IgM Fab mu fragment (myeloma)-Biotin conjugate (isotype control)
20007-4-B 100 ug
EUR 225
Human IgG-Fc fragment (97%, low endotoxin, azide free)
20007-F-LE-100 100 ug
EUR 164
Human IgG-Fc fragment-Biotin (97%, low endotoxin, azide free)
20007-F-LE-BTN 100 ug
EUR 286
Human IgG1-Biotin Conjugate (-ve control)
20007-G1-B 100 ug
EUR 164
Recombinant (HEK) Human IgG1 Fc (103Cys/Ser, 99-330aa, His-tag, >95%, low endotoxin)
20007-G1-FC 50 ug
EUR 286
Human IgG2-Biotin Conjugate
20007-G2-B 100 ug
EUR 164
Recombinant (HEK) Human IgG2-Fc (257Ser/Ala, 99-327aa, His-tag, >95%, low endotoxin)
20007-G2-FC 50 ug
EUR 286
Human IgG3-Biotin Conjugate
20007-G3-B 100 ug
EUR 164
Recombinant (HEK) Human IgG3-Fc (99-377aa, His-tag, >95%, low endotoxin)
20007-G3-FC 50 ug
EUR 286
Human IgG4-Biotin Conjugate
20007-G4-B 100 ug
EUR 164
Recombinant (HEK) Human IgG4-Fc (108Ser/Pro, 99-327aa, His-tag, >95%, low endotoxin)
20007-G4-FC 50 ug
EUR 286
Mouse IgG F(ab')2 fragment, purified
20008-1-Fab2 1 mg
EUR 202
Mouse IgG (>97%, ELISA/RIA/Diagnostic Grade; -ve mouse viruses & Protein A/G free)
20008-1-WA 1 mg
EUR 164
Mouse IgG-Agarose conjugate (aff matrix)
20008-AS-1 0.5 ml
EUR 164
Mouse IgG-Biotin conjugate (isotype control)
20008-B 50 Tests
EUR 164
Mouse IgG-Biotin conjugate (isotype control)
20008-BT-1 1 mg
EUR 164
Mouse IgG Fab-Biotin conjugate (isotype control)
20008-Fab-B 100 ug
EUR 164
Mouse IgG F(ab')2-Biotin conjugate (isotype control)
20008-Fab2-B 100 ug
EUR 164
Mouse IgG F(ab')2-FITC conjugate (isotype control)
20008-Fab2-F 100 ug
EUR 164
Mouse IgG F(ab')2-HRP conjugate (isotype control)
20008-Fab2-HP 100 ug
EUR 164
Mouse IgG F(c)-Biotin conjugate (isotype control)
20008-Fc-B 100 ug
EUR 164
Rabbit IgG Fab-Biotin Conjugate, purified
20009-1-FAB-B 0.1 mg
EUR 202
Rabbit IgG F(ab')2 fragment, purified
20009-1-FAB2 1 mg
EUR 202
Recombinant (HEK) Rabbit IgG (Fc) (101-323aa, 185Thr/Asn284Ser, ~33 kda, >95%, Low Endotoxin)
20009-1-R-FC 50 ug
EUR 286
Rabbit IgM-Biotin Conjugate (isotype control, non-immune), purified
20009-2-1-B 0.1 mg
EUR 225
Rabbit IgA-Biotin Conjugate (isotype control, non-immune), purified
20009-3-1-B 0.1 mg
EUR 408
Rabbit IgG-Agarose conjugate
20009-AS-1 0.5 ml
EUR 164
Rabbit IgG (Whole, non-immune)-Biotin conjugate
20009-B 100 ug
EUR 164
Rabbit IgG-biotinylated (isotype control)
20009-BT-1 1 mg
EUR 286
Rabbit IgG F(ab')2-biotin conjugate, isotype control
20009-FAb2-B 100 ug
EUR 164
Rabbit IgG F(ab')2-FITC conjugate, isotype control
20009-FAb2-F 100 ug
EUR 164
Rabbit IgG F(ab')2-HRP conjugate, isotype control
20009-FAb2-HP 100 ug
EUR 164
Rabbit IgG F(ab')2-R-PE conjugate, isotype control
20009-FAb2-PE 100 tests
EUR 225
Rabbit IgG F(c)-biotin conjugate, isotype control
20009-Fc-B 100 ug
EUR 164
Rabbit IgG, purified (serum non-immune, isotype control), without azide, powder
20009-WA-1 1 mg
EUR 141
Rabbit IgG, purified (serum non-immune, isotype control), without azide, powder
20009-WA-5 5 mg
EUR 286
Human Insulin Antibody
20010-05011 150 ug
EUR 217
Human Insulin Antibody (Biotin Conjugate)
20010-05021 150 ug
EUR 276
Chicken IgG-Biotin conjugate (isotype control, non-immune), purified
20010-1-B 0.5 mg
EUR 225
Chicken IgM-Biotin conjugate (isotype control, non-immune) purified
20010-2-1-b 0.1 mg
EUR 225
Chicken IgG-Fc-Biotin conjugate (isotype control, non-immune), purified
20010-3-B 0.1 mg
EUR 225
Human Synaptobrevin-2 Antibody
20011-05011 150 ug
EUR 217
Human Synaptobrevin-2 Antibody (Biotin Conjugate)
20011-05021 150 ug
EUR 276
Human Synaptobrevin-2 AssayLite Antibody (FITC Conjugate)
20011-05041 150 ug
EUR 428
Human Synaptobrevin-2 AssayLite Antibody (RPE Conjugate)
20011-05051 150 ug
EUR 428
Human Synaptobrevin-2 AssayLite Antibody (APC Conjugate)
20011-05061 150 ug
EUR 428
Human Synaptobrevin-2 AssayLite Antibody (PerCP Conjugate)
20011-05071 150 ug
EUR 471
Goat IgG FAB fragment-Biotin conjugate
20011-1-FAB-B 100 tests
EUR 225
Goat IgG F(ab')2 fragment, purified
20011-1-FAB2 1 mg
EUR 164
Goat IgG-Agarose conjugate(aff matrix)
20011-AS-1 0.5 ml
EUR 164
Goat IgG (Whole, non-immune)-Biotin conjugate
20011-B 100 ug
EUR 164
Goat IgG F(ab')2 fragment-Biotin conjugate
20011-FAB2-B 100 tests
EUR 202

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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