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A novel simple assay system to quantify the percent HCV-RNA levels of NS5A Y93H mutant strains and Y93 wild-type strains relative to the total HCV-RNA levels to determine the indication for antiviral therapy with NS5A inhibitors.

Uchida Y, Kouyama J, Naiki K, Mochida S - PLoS ONE (2014)

Bottom Line: The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing.A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established.This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.

ABSTRACT

Aim: Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.

Methods: Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.

Results: Both the wild-type and mutant strains were quantified, with a threshold of 4.0 Log copies/mL, in 295 of the 332 patients (88.9%), and the percentage of the mutant strains relative to the total HCV-RNA level in the serum was calculated. The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing. Both wild-type and mutant strains were detected in the remaining 35 patients (11.9%), at levels between 1% and 99%, despite the mutant strains having been undetectable by direct sequencing in 11 patients with percentages of these strains of less than 25%.

Conclusion: A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established. This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.

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Related in: MedlinePlus

Real-Time PCR Analysis Using Mixtures of Synthesized Oligonucleoside Following Adjustment to Different Percent Volumes of Both Oligonucleosides.SYN-NS5A_Hcac/SYN-NS5A_Ycac and SYN-NS5A_Hcgc/SYN-NS5A_Ycgc were used as the mixtures and the percent volumes of both oligonucleosides were 0%, 5%, 10%, 30%, 50%, 70%, 90% or 100%. Ycac/Hcac or Ycgc/Hcgc was used as the cycling probe. The numbers on the left indicate the percent volumes of the SYN-NS5A_Hcac/SYN-NS5A_ Hcgc in each sample.
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pone-0112647-g002: Real-Time PCR Analysis Using Mixtures of Synthesized Oligonucleoside Following Adjustment to Different Percent Volumes of Both Oligonucleosides.SYN-NS5A_Hcac/SYN-NS5A_Ycac and SYN-NS5A_Hcgc/SYN-NS5A_Ycgc were used as the mixtures and the percent volumes of both oligonucleosides were 0%, 5%, 10%, 30%, 50%, 70%, 90% or 100%. Ycac/Hcac or Ycgc/Hcgc was used as the cycling probe. The numbers on the left indicate the percent volumes of the SYN-NS5A_Hcac/SYN-NS5A_ Hcgc in each sample.

Mentions: Next, real-time PCR was performed with mixtures of the synthesized oligonucleosides, SYN-NS5A_Ycac and SYN-NS5A_Hcac or SYN-NS5A_Ycgc and SYN-NS5A_Hcgc, adjusted to different percent volumes of both oligonucleosides: 0%, 5%, 10%, 30%, 50%, 70%, 90% and 100%. The results for the SYN-NS5A_Ycac/SYN-NS5A_Hcac mixtures after real-time PCR with Ycac and Hcac as the cycling probes are shown in Figure 2. The PCR products of SYN-NS5A_Hcac and SYN-NS5A_Ycac were separately quantified by real-time PCR in each mixture with different proportions of both oligonucleosides; the error range in each mixture was less than 10% (Table 3). Similar results were obtained in experiments using SYN-NS5A_Hcgc and SYN-NS5A_Yagc as the oligonucleosides and Hcgc and Ycgc as the cycling probes (Table 3).


A novel simple assay system to quantify the percent HCV-RNA levels of NS5A Y93H mutant strains and Y93 wild-type strains relative to the total HCV-RNA levels to determine the indication for antiviral therapy with NS5A inhibitors.

Uchida Y, Kouyama J, Naiki K, Mochida S - PLoS ONE (2014)

Real-Time PCR Analysis Using Mixtures of Synthesized Oligonucleoside Following Adjustment to Different Percent Volumes of Both Oligonucleosides.SYN-NS5A_Hcac/SYN-NS5A_Ycac and SYN-NS5A_Hcgc/SYN-NS5A_Ycgc were used as the mixtures and the percent volumes of both oligonucleosides were 0%, 5%, 10%, 30%, 50%, 70%, 90% or 100%. Ycac/Hcac or Ycgc/Hcgc was used as the cycling probe. The numbers on the left indicate the percent volumes of the SYN-NS5A_Hcac/SYN-NS5A_ Hcgc in each sample.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232421&req=5

pone-0112647-g002: Real-Time PCR Analysis Using Mixtures of Synthesized Oligonucleoside Following Adjustment to Different Percent Volumes of Both Oligonucleosides.SYN-NS5A_Hcac/SYN-NS5A_Ycac and SYN-NS5A_Hcgc/SYN-NS5A_Ycgc were used as the mixtures and the percent volumes of both oligonucleosides were 0%, 5%, 10%, 30%, 50%, 70%, 90% or 100%. Ycac/Hcac or Ycgc/Hcgc was used as the cycling probe. The numbers on the left indicate the percent volumes of the SYN-NS5A_Hcac/SYN-NS5A_ Hcgc in each sample.
Mentions: Next, real-time PCR was performed with mixtures of the synthesized oligonucleosides, SYN-NS5A_Ycac and SYN-NS5A_Hcac or SYN-NS5A_Ycgc and SYN-NS5A_Hcgc, adjusted to different percent volumes of both oligonucleosides: 0%, 5%, 10%, 30%, 50%, 70%, 90% and 100%. The results for the SYN-NS5A_Ycac/SYN-NS5A_Hcac mixtures after real-time PCR with Ycac and Hcac as the cycling probes are shown in Figure 2. The PCR products of SYN-NS5A_Hcac and SYN-NS5A_Ycac were separately quantified by real-time PCR in each mixture with different proportions of both oligonucleosides; the error range in each mixture was less than 10% (Table 3). Similar results were obtained in experiments using SYN-NS5A_Hcgc and SYN-NS5A_Yagc as the oligonucleosides and Hcgc and Ycgc as the cycling probes (Table 3).

Bottom Line: The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing.A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established.This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.

ABSTRACT

Aim: Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.

Methods: Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.

Results: Both the wild-type and mutant strains were quantified, with a threshold of 4.0 Log copies/mL, in 295 of the 332 patients (88.9%), and the percentage of the mutant strains relative to the total HCV-RNA level in the serum was calculated. The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing. Both wild-type and mutant strains were detected in the remaining 35 patients (11.9%), at levels between 1% and 99%, despite the mutant strains having been undetectable by direct sequencing in 11 patients with percentages of these strains of less than 25%.

Conclusion: A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established. This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.

Show MeSH
Related in: MedlinePlus