Optimization of PCR Conditions for Amplification of GC-RichEGFRPromoter Sequence

Obradovic, Jasmina; Jurišić, Vladimir; Tos̆ić, Nataša; Mrdjanović, Jasminka; Perin, Branislav; Pavlović, Sonja; Djordjević, Nataša

doi:10.1002/jcla.21632

Cited by 31 publications

(25 citation statements)

References 44 publications

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“…The 3 Giardia -positive control samples that could not be picked up by the assay were assumed to be of low parasite loads. The exhibited outstanding assay specificity may be related to the high specificity of primers, the usage of the touchdown protocol, and inclusion of 1% DMSO in the reaction, as previously stated [ 29 , 30 ]. Subsequently, the assay was validated on a panel of stool samples with blinded results.…”

Section: Discussionmentioning

confidence: 94%

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

2016

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Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

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Section: Discussionmentioning

confidence: 94%

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

2016

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“…To avoid potential problems with PCR optimization, we performed genotyping using a ready‐to‐use assay (cat. no.…”

Section: Methodsmentioning

confidence: 99%

Possible association between actinic keratosis and the rs7208422 (c.917A→T, p.N306l) polymorphism of theEVER2gene in patients without epidermodysplasia verruciformis

et al. 2014

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“…EGFR promoter activation requires transcription factor Sp1, for which multiple binding sites were discovered [ 31 , 33 – 37 ]. EGFR transcription is upregulated by at least three enhancers that act cooperatively: two of them localized upstream, i.e., near the start transcription site, and the third one in introne [ 38 – 40 ].…”

Section: Egfr Gene Regulationmentioning

confidence: 99%

Epidermal Growth Factor Receptor Gene in Non-Small-Cell Lung Cancer: The Importance of Promoter Polymorphism Investigation

Jurišić

Obradovic

Pavlović

et al. 2018

Analytical Cellular Pathology

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Recently, epidermal growth factor receptor (EGFR) was a key molecule in investigation of lung cancer, and it was a target for a new therapeutic strategy, based on molecular analyses. In this review, we have summarized some issues considering the role of EGFR in lung cancer, its coding gene, and its promoter gene polymorphisms (SNPs) -216G/T and -191C/A in non-small-cell lung cancer (NSCLC). The position of the SNPs indicates their significant role in EGFR regulation. The accumulation of knowledge regarding SNPs lately suggests their significant and important role in the onset of carcinogenesis, the prediction of the onset of metastases, the response to therapy with TKI inhibitors, and the onset of toxic effects of the applied therapy. Based on this, we suggest further studies of the relationship of clinical significance to SNPs in patients with lung tumors.

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Optimization of PCR Conditions for Amplification of GC-RichEGFRPromoter Sequence

Abstract: In conclusion, EGFR promoter region is a difficult PCR target, but it could be amplified after optimization of MgCl2 concentration and annealing temperature in the presence of DMSO and the DNA template of acceptable concentration.

Cited by 31 publications

References 44 publications

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic <i>Giardia lamblia</i> Infection in a Resource-Poor Setting

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic <i>Giardia lamblia</i> Infection in a Resource-Poor Setting

Possible association between actinic keratosis and the rs7208422 (c.917A→T, p.N306l) polymorphism of theEVER2gene in patients without epidermodysplasia verruciformis

Epidermal Growth Factor Receptor Gene in Non-Small-Cell Lung Cancer: The Importance of Promoter Polymorphism Investigation

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