3 research outputs found
Comparison of sensitivity of nested PCR and quantitative PCR in Bcr-Abl p210 transcript detection in chronic myelogenous leukemia
Uvod: Za otkrivanje minimalne ostatne bolesti u bolesnika s kroniÄnom mijeloiÄnom leukemijom (KML) koji su postigli potpunu kliniÄku remisiju i potpun citogenetski odgovor može se primijeniti ugnježÄena PCR (engl. nestedPCR, nPCR) i kvantitativna PCR u stvarnom vremenu (engl. quantitative real-time PCR, qPCR). Cilj lijeÄenja je postizanje molekularne remisije, pa postizanje visoke osjetljivosti molekularne pretrage ima presudnu ulogu i kliniÄku primjenu. Usporedili smo razinu osjetljivosti nPCR i qPCR u otkrivanju BCR-ABL p210 prijepisa u modelu razrjeÄenja Bcr/Abl-pozitivnih stanica.
Materijal i metode: Za odreÄivanje razine osjetljivosti naÄinjena su serijska razrjeÄenja staniÄne linije K562 (Bcr-Abl pozitivne) sa staniÄnom linijom NB4 (Bcr-Abl negativnom) (raspon razrjeÄenja pozitivnih stanica: 10-3-10-7). Izolirani uzorci RNA prepisani su u cDNA i testirani na p210 prijepis pomoÄu nPCR i qPCR. Objema metodama takoÄer su ispitani uzorci koÅ”tane srži i periferne krvi dvoje bolesnika s KML na terapiji imatinib-mesilatom.
Rezultati: U testu staniÄnog razrjeÄenja nPCR je pokazala osjetljivost za otkrivanje Bcr-Abl pozitivnih stanica od 10-5, dok je qPCR pokazala osjetljivost od 10-6. Obje su metode otkrile p210 prijepise u uzorcima koÅ”tane srži bolesnika s KML. MeÄutim, qPCR je uz to otkrila prijepise i u uzorcima periferne krvi, no uz nižu razinu prijepisa u usporedbi s uzorcima koÅ”tane srži.
ZakljuÄak: Iako se Äesto navodi kako je nPCR za otprilike 1 log osjetljivija od qPCR, u naÅ”em pokusu s razrjeÄenjem na biljeg pozitivne staniÄne linije K562 dokumentirali smo viÅ”u osjetljivost za standardiziranu metodu qPCR, koja je razvijena za potrebe Europskog programa za borbu protiv karcinoma.Background: For minimal residual disease detection in chronic myelogenous leukemia (CML) patients who have achieved complete clinical remission and complete cytogenetic response, nested PCR (nPCR) and quantitative realtime PCR (qPCR) can be used. Achieving of molecular remission is the goal of therapy, so it is of critical importance and clinical utility to obtain high sensitivity of molecular testing. We compared the level of sensitivity of nPCR and qPCR in the detection of BCR-ABL p210 transcripts in a Bcr/Abl-positive cell dilution model.
Materials and Methods: For determination of sensitivity level, serial dilutions of K562 cell line (Bcr-Abl-positive) in NB4cell line (Bcr-Abl-negative) were made (range of dilution of positive cells: 10-3-10-7). Isolated RNA samples were transcribed into cDNA and tested for p210 transcript by nPCR and qPCR. Bone marrow and peripheral blood samples of two CML patients on imatinib mesylate therapy were also tested by both methods.
Results: In the cell dilution test, nPCR showed sensitivity for detecting Bcr-Abl positive cell of 10-5, and qPCR showed a sensitivity of 10-6. Both methods detected p210 transcripts in bone marrow samples of CML patients. However, qPCR also detected transcripts in peripheral blood samples, with a lower transcript level in comparison to bone marrow samples.
Conclusion: Although frequently quoted as nPCR being by approximately 1 log more sensitive than qPCR, in our marker-positive cell line K562 dilution experiment we documented higher sensitivity of the standardized Europe Against Cancer Program developed qPCR method