Analysis of the qPCR Assay Efficacy as Molecular Diagnostic in Patients with Chemotherapy and/or Antibiotic Therapy
Braz. arch. biol. technol
;
64: e21190423, 2021. tab, graf
Article
in English
| LILACS
| ID: biblio-1285548
ABSTRACT
Abstract High sensitivity of qPCR assay can be compromised by the presence of PCR inhibitors in samples analyzed. The aim of this study was to analyze the RT-qPCR assay efficiency considering the RNA quality/quantity and the presence of PCR inhibitors in patients with chemotherapy and/or antibiotic therapy. We analyzed 60 samples using RT-qPCR from individuals suspected of leukemia and 44 samples were quantified by fluorimetry and spectrophotometry. The efficiency of the RT-qPCR assay was evaluated comparing the threshold cycle (Ct) from tested samples and the standard curve. The 260/280 and 260/230 ratios, the presence of PCR inhibitors and the amount of sample (ng) used in the RT-qPCR reaction can be associated with 56.8% (R²=0.56, p<0.05) in the Ct obtained. The decrease of the RT-qPCR efficiency can be explained in 42,8% due to the variation of the 260/280 ratio (R²=0.42,p<0.05). The presence of antibiotics in the blood sample can be associated in 11.3% with the variability of 260/280 ratio (R²=0.11,p<0.05). Presence of chemotherapeutic drugs in the blood sample was not correlated with Ct variation (p=0.17). The spectrophotometer determines a RNA quantification with 2.2 times higher than the fluorimeter (t=2.2, p=0,03) and this difference is correlated with the 260/280 ratio (R²=0.36, p<0.05). Samples with low purity had a reduction in the qPCR efficiency, although we did not observe false results.
Full text:
Available
Index:
LILACS (Americas)
Main subject:
Polymerase Chain Reaction
/
Nucleic Acid Synthesis Inhibitors
/
Molecular Diagnostic Techniques
Type of study:
Diagnostic study
Limits:
Humans
Language:
English
Journal:
Braz. arch. biol. technol
Journal subject:
Biology
Year:
2021
Type:
Article
Affiliation country:
Brazil
Institution/Affiliation country:
UNIFAMINAS University/BR
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