Monitoring HCV RNA viral load by locked nucleic acid molecular beacons real time PCR.
Locked nucleic acids (LNA) based real time PCR was used in particular situations where there are difficulties in primer design due to sequence complexity. In this study a new real time RT-PCR assay was developed using LNA modified primers and LNA molecular beacon probes to monitor hepatitis C virus (HCV) viral load in plasma and serum samples. The technique did not suffer from an heterogeneity of the HCV genome and, in addition, an internal RNA control was amplified in the same reaction tube with different short primers and beacon probe. Due to the short consensus LNA primers length, the PCR efficiency was close to 100% with no formation of hairpin loop structures. In summary a new LNA molecular beacon based real time RT-PCR assay was used successfully to measure quantitatively the total level of HCV RNA in both experimental and clinical specimens. The high sensitivity (50 IU/ml), the wide range of genotype detection, increased specificity and robustness obtained with this test are particularly useful for screening large number of specimens and measuring viral loads to monitor the progress of the disease.
Morandi
Luca
L
Department of Oncology, Section of Pathology, University of Bologna, Ospedale Bellaria, Bologna, Italy.
Ferrari
Daniela
D
Lombardo
Claudia
C
Pession
Annalisa
A
Tallini
Giovanni
G
D020121:5' Untranslated Regions; D017931:DNA Primers; D015342:DNA Probes; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load
D020121:5' Untranslated Regions; D017931:DNA Primers; D015342:DNA Probes; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012367:RNA, Viral; C477371:locked nucleic acid