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Medicina
Croatian Medical Association - Rijeka branch
ISSN: 0025-7729
Vol. 44, No. 3-4, 2008, pp. 297-302
Bioline Code: me08037
Full paper language: Croatian
Document type: Research Article
Document available free of charge

Medicina, Vol. 44, No. 3-4, 2008, pp. 297-302

 en Influence of PCR inhibitors on molecular diagnosis of pneumococcal pneumonia
Mareković, Ivana; Plečko, Vanda; Boras, Zagorka; Pavlović, Ladislav; Budimir, Ana; Bošnjak, Zrinka; Kamauf-Balabanić, Blanka; Marinković, Sonja; Žele-Starčević, Lidija & Kalenić, Smilja

Abstract

Aim: To determine if inhibitors in blood and respiratory specimens influence the polymerase chain reaction (PCR) results for Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). Patients and methods: The study included 80 patients with CAP. In addition to conventional microbiological methods, PCR for fragment of pneumolysin gene in serum (N=77) and BAL (N=19) specimens was also performed. The presence of PCR inhibitors in specimens was determined with spiking and inhibitors were removed by diluting the specimens. Results: With all methods, S. pneumoniae was detected in 25 (31,3 %) of patients with CAP. PCR results in 77 serum specimens were negative. PCR inhibitors were detected in one serum specimen. BAL specimens were PCR-positive in three patients and there were no inhibitors detected. Discussion: When planning and performing PCR on clinical specimens, PCR inhibitors should be considered as a possible cause of false-negative results. Several procedures are used to remove PCR inhibitors from clinical specimens or to diminish their influence (extraction method, optimizing DNA amplification, diluting of specimens). Presence of inhibitors can be determined by adding of an internal control to each specimen or with spiking. Conclusions: In this study the presence of inhibitors was determined in only one serum specimen. It can be concluded that PCR inhibitors in serum and BAL specimens do not influence the PCR results for S. pneumoniae in patients with CAP.

Keywords
blood, bronchoalveolar lavage (BAL), community-acquired pneumonia, inhibition, PCR

 
 hr Utjecaj PCR inhibitora na molekularnu dijagnostiku pneumokokne pneumonije
Mareković, Ivana; Plečko, Vanda; Boras, Zagorka; Pavlović, Ladislav; Budimir, Ana; Bošnjak, Zrinka; Kamauf-Balabanić, Blanka; Marinković, Sonja; Žele-Starčević, Lidija & Kalenić, Smilja


Cilj: Utvrditi utjecaj inhibitora prisutnih u uzorcima krvi i njenih frakcija te u uzorcima iz respiratornog sustava na rezultate PCR-a za Streptococcus pneumoniae u bolesnika s izvanbolničkom pneumonijom. Ispitanici i metode: U istraživanje je bilo uključeno 80 ispitanika s izvanbolničkom pneumonijom u kojih je, osim konvencionalnih mikrobioloških pretraga, napravljena i lančana reakcija polimeraze (engl. polymerase chain reaction, PCR) za fragment gena pneumolizina na uzorcima seruma (N=77) i bronhoalveolarnih ispiraka (BAL) (N=19). U uzorcima je utvrđivana prisutnost PCR inhibitora, a za njihovo uklanjanje korišteno je razrjeđivanje uzoraka. Rezultati: Primijenjenim mikrobiološkim metodama S. pneumoniae dokazan je kod ukupno 25 (31,3 %) ispitanika s izvanbolničkom pneumonijom. PCR rezultati u svih 77 uzoraka seruma bili su negativni, a prisutnost PCR inhibitora utvrđena je u jednom uzorku seruma. PCR je u uzorcima BAL-a bio pozitivan kod tri ispitanika, a inhibitori nisu utvrđeni niti u jednom uzorku BAL-a. Rasprava: Pri planiranju i izvođenju PCR-a na kliničkim uzorcima treba misliti na moguću prisutnost PCR inhibitora koji mogu biti uzrok lažno-negativnih rezultata. Određenim postupcima PCR inhibitori mogu se ukloniti iz kliničkih uzoraka ili se njihovo djelovanje može umanjiti (metoda izolacije DNA, optimizacija uvjeta umnažanja, razrjeđivanje uzoraka). Prisutnost inhibitora u uzorku može se utvrditi dodavanjem interne kontrole u svaki uzorak ili pomoću spikinga. Zaključak: S obzirom na to da je u ovom istraživanju prisutnost inhibitora utvrđena samo u jednom uzorku seruma, može se zaključiti da PCR inhibitori u uzorcima seruma i BAL-a ne utječu na rezultate PCR-a za S. pneumoniae u bolesnika s izvanbolničkom pneumonijom.


bronhoalveolarni ispirak (BAL), inhibicija, izvanbolnička pneumonija, krv, PCR

 
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