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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 a Synthesized Oligonucleoside Following Adjustment for a 100-fold Dilution Series from 1×102 to 1×108 copies/mL.(A) Amplification plots using SYN-NS5A_Ycac. (B) The standard curve for SYN-NS5A_Ycac generated from the amplification plots displayed in Fig 1A. The correlation coefficient was 0.998. (C)Amplification plots using SYN-NS5A_Hcac. (D) The standard curve for SYN-NS5A_Hcac. The correlation coefficient was 0.998. All figures were generated by the ABI Psism 7900 Sequence Detection Software (SDS), version 2.4.
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pone-0112647-g001: Real-Time PCR Analysis Using a Synthesized Oligonucleoside Following Adjustment for a 100-fold Dilution Series from 1×102 to 1×108 copies/mL.(A) Amplification plots using SYN-NS5A_Ycac. (B) The standard curve for SYN-NS5A_Ycac generated from the amplification plots displayed in Fig 1A. The correlation coefficient was 0.998. (C)Amplification plots using SYN-NS5A_Hcac. (D) The standard curve for SYN-NS5A_Hcac. The correlation coefficient was 0.998. All figures were generated by the ABI Psism 7900 Sequence Detection Software (SDS), version 2.4.

Mentions: First of all, real-time PCR was carried out with the synthesized oligonucleotides following adjustment for a 100-fold dilution series. As shown in Figure 1, the PCR products of SYN-NS5A_Ycac and SYN-NS5A_Hcac were quantified depending on their copy levels with Ycnc and Hcnc, respectively, as cycling probes (from 1×102 to 1×108 copies/mL), while no products were detected when the cycling probes were used in reverse, that is, Hcnc and Ycnc for SYN-NS5A_Ycac and SYN-NS5A_Hcac, respectively. Similar results were also obtained in the experiments using SYN-NS5A_Ycgc and SYN-NS5A_Hcgc as the synthesized oligonucleotides.


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 a Synthesized Oligonucleoside Following Adjustment for a 100-fold Dilution Series from 1×102 to 1×108 copies/mL.(A) Amplification plots using SYN-NS5A_Ycac. (B) The standard curve for SYN-NS5A_Ycac generated from the amplification plots displayed in Fig 1A. The correlation coefficient was 0.998. (C)Amplification plots using SYN-NS5A_Hcac. (D) The standard curve for SYN-NS5A_Hcac. The correlation coefficient was 0.998. All figures were generated by the ABI Psism 7900 Sequence Detection Software (SDS), version 2.4.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112647-g001: Real-Time PCR Analysis Using a Synthesized Oligonucleoside Following Adjustment for a 100-fold Dilution Series from 1×102 to 1×108 copies/mL.(A) Amplification plots using SYN-NS5A_Ycac. (B) The standard curve for SYN-NS5A_Ycac generated from the amplification plots displayed in Fig 1A. The correlation coefficient was 0.998. (C)Amplification plots using SYN-NS5A_Hcac. (D) The standard curve for SYN-NS5A_Hcac. The correlation coefficient was 0.998. All figures were generated by the ABI Psism 7900 Sequence Detection Software (SDS), version 2.4.
Mentions: First of all, real-time PCR was carried out with the synthesized oligonucleotides following adjustment for a 100-fold dilution series. As shown in Figure 1, the PCR products of SYN-NS5A_Ycac and SYN-NS5A_Hcac were quantified depending on their copy levels with Ycnc and Hcnc, respectively, as cycling probes (from 1×102 to 1×108 copies/mL), while no products were detected when the cycling probes were used in reverse, that is, Hcnc and Ycnc for SYN-NS5A_Ycac and SYN-NS5A_Hcac, respectively. Similar results were also obtained in the experiments using SYN-NS5A_Ycgc and SYN-NS5A_Hcgc as the synthesized oligonucleotides.

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