[CMV DNA detection in plasma using real-time PCR based on the SYBR-Green I dye method].
OBJECTIVE
The aim of this study is to assess a real-time PCR technique on the LightCycler 2.0 with SYBR-Green I detection as compared to another real-time PCR method based on detection with FRET (fluorescence resonance energy transfer) probes for the quantification of CMV DNA.


METHODS
The two real-time PCR methods were used to test plasma samples from immunocompromised patients with clinically suspected CMV disease, patients under follow-up without symptoms, and healthy adults. A standard curve for quantitative analysis by the SYBR-Green I method was performed with 10-fold diluted solutions of DNA from the CMV Towne strain (ATCC VR-977) cultured in MRC-5 monolayer. In addition, frozen samples from patients positive for CMV pp65 antigenemia were also analyzed and results compared using the two real time PCR methods.


RESULTS
The real-time PCR technique using SYBR-Green I on the LightCycler 2.0 was a highly specific, fast, simple and reliable test to quantify CMV; moreover, it was cost-effective.


CONCLUSIONS
Quantification of CMV DNA in plasma using this sensitive, fast, low-cost method was advantageous for the diagnosis and follow up of patients with opportunistic CMV infection, which are increasingly more frequent in our daily hospital clinical practice.
Varela-Ledo
Eduardo
E
Servicio de Microbiología, Complejo Hospitalario Universitario de Santiago de Compostela, España. eduardo.varela.ledo@sergas.es
Romero-Yuste
Susana
S
Ordóñez-Barbosa
Patricia
P
Romero-Jung
Patricia
P
Prieto-Rodríguez
Elisabeth
E
Aguilera-Guirao
Antonio
A
Regueiro-García
Benito
B
D017088:AIDS-Related Opportunistic Infections / Q000097:blood / Q000175:diagnosis / Q000821:virology; D000328:Adult; D000956:Antigens, Viral / Q000097:blood*; D052160:Benzothiazoles; D016026:Bone Marrow Transplantation; D002460:Cell Line; D002648:Child; D003199:Computer Systems / Q000191:economics; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000097:blood* / Q000175:diagnosis / Q000821:virology; D004279:DNA, Viral / Q000097:blood*; D003959:Diamines; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D016867:Immunocompromised Host; D007231:Infant, Newborn; D009369:Neoplasms / Q000150:complications; D009504:Neutrophils / Q000821:virology; D009894:Opportunistic Infections / Q000097:blood* / Q000175:diagnosis / Q000821:virology; D009930:Organic Chemicals / Q000032:analysis*; D010750:Phosphoproteins / Q000097:blood; D016133:Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D011183:Postoperative Complications / Q000097:blood / Q000175:diagnosis / Q000821:virology; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000097:blood; D014766:Viremia / Q000097:blood* / Q000175:diagnosis / Q000821:virology
D000956:Antigens, Viral; D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D010750:Phosphoproteins; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; C098022:SYBR Green I