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Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

Yoshimi S, Ochi H, Murakami E, Uchida T, Kan H, Akamatsu S, Hayes CN, Abe H, Miki D, Hiraga N, Imamura M, Aikata H, Chayama K - PLoS ONE (2015)

Bottom Line: Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed.Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain.Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.

ABSTRACT
Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

No MeSH data available.


Related in: MedlinePlus

Schematic flow diagram representing a method of nested-PCR followed by Invader assay.(A) The initial PCR was performed to amplify a fragment of 386 bp length containing a part of the NS5A region from cDNA reverse-transcribed from HCV-RNA which was extracted from the serum of a patient. (B) An aliquot of the inital PCR product was used for the second PCR to amplify a 308 bp fragment. The second PCR product was diluted with water and subjected to the Invader assay. (C) Invader oligonucleotide and allele-specific probes anneal with target to form one base overlap. When the base is complementary to the opposing base in the allele-specific probe, cleavase recognizes the structure and releases 5’ flap. The released flap anneals to a FRET probe. The second cleavage reaction releases fluorophore resulting in the generation of a fluorescent signal. The Invader assay was done in triplicate. (D) Three types of standard nucleotide (Std-YY, Std-YH, and Std-HH) were prepared, which includes binary target sequences, corresponding to wild (Y) or mutant (H) variants, and annealing sites with the internal primers at each end. Tenfold serial dilusions of each standard were subjected to the second PCR. (E) The Invader assay for each standard was also performed in triplicate.
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pone.0130022.g003: Schematic flow diagram representing a method of nested-PCR followed by Invader assay.(A) The initial PCR was performed to amplify a fragment of 386 bp length containing a part of the NS5A region from cDNA reverse-transcribed from HCV-RNA which was extracted from the serum of a patient. (B) An aliquot of the inital PCR product was used for the second PCR to amplify a 308 bp fragment. The second PCR product was diluted with water and subjected to the Invader assay. (C) Invader oligonucleotide and allele-specific probes anneal with target to form one base overlap. When the base is complementary to the opposing base in the allele-specific probe, cleavase recognizes the structure and releases 5’ flap. The released flap anneals to a FRET probe. The second cleavage reaction releases fluorophore resulting in the generation of a fluorescent signal. The Invader assay was done in triplicate. (D) Three types of standard nucleotide (Std-YY, Std-YH, and Std-HH) were prepared, which includes binary target sequences, corresponding to wild (Y) or mutant (H) variants, and annealing sites with the internal primers at each end. Tenfold serial dilusions of each standard were subjected to the second PCR. (E) The Invader assay for each standard was also performed in triplicate.

Mentions: Three nucleotide sequences, which also contained several degenerate sites, were designed for the Invader assay (Table 1). For the calibration of the proportion of mutant variants, three types of standard nucleotide were prepared: Std-YY, Std-YH, and Std-HH (Table 1). Each sequence includes binary target sequences, corresponding to wild (Y) or mutant (H) variants, and also includes annealing sites with the internal primers at each end. An additional four nucleotides were inserted between the binary target sequences to avoid interference. Dilution series of these standards were assayed in triplicate with each assay. For Invader assays, 384-well reaction plates were used with the ABI Prism 7900 HT sequence detection system (Life Technologies, Foster City, CA, USA). The plate was incubated isothermally at 63°C and fluorescence intensities were detected every 2 min for 40 min or until non-specific fluorescence [21] was detected. Each sample was tested in triplicate in the same plate. A flow diagram representing a method of nested-PCR followed by Invader assay is depicted in Fig 3.


Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

Yoshimi S, Ochi H, Murakami E, Uchida T, Kan H, Akamatsu S, Hayes CN, Abe H, Miki D, Hiraga N, Imamura M, Aikata H, Chayama K - PLoS ONE (2015)

Schematic flow diagram representing a method of nested-PCR followed by Invader assay.(A) The initial PCR was performed to amplify a fragment of 386 bp length containing a part of the NS5A region from cDNA reverse-transcribed from HCV-RNA which was extracted from the serum of a patient. (B) An aliquot of the inital PCR product was used for the second PCR to amplify a 308 bp fragment. The second PCR product was diluted with water and subjected to the Invader assay. (C) Invader oligonucleotide and allele-specific probes anneal with target to form one base overlap. When the base is complementary to the opposing base in the allele-specific probe, cleavase recognizes the structure and releases 5’ flap. The released flap anneals to a FRET probe. The second cleavage reaction releases fluorophore resulting in the generation of a fluorescent signal. The Invader assay was done in triplicate. (D) Three types of standard nucleotide (Std-YY, Std-YH, and Std-HH) were prepared, which includes binary target sequences, corresponding to wild (Y) or mutant (H) variants, and annealing sites with the internal primers at each end. Tenfold serial dilusions of each standard were subjected to the second PCR. (E) The Invader assay for each standard was also performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4470996&req=5

pone.0130022.g003: Schematic flow diagram representing a method of nested-PCR followed by Invader assay.(A) The initial PCR was performed to amplify a fragment of 386 bp length containing a part of the NS5A region from cDNA reverse-transcribed from HCV-RNA which was extracted from the serum of a patient. (B) An aliquot of the inital PCR product was used for the second PCR to amplify a 308 bp fragment. The second PCR product was diluted with water and subjected to the Invader assay. (C) Invader oligonucleotide and allele-specific probes anneal with target to form one base overlap. When the base is complementary to the opposing base in the allele-specific probe, cleavase recognizes the structure and releases 5’ flap. The released flap anneals to a FRET probe. The second cleavage reaction releases fluorophore resulting in the generation of a fluorescent signal. The Invader assay was done in triplicate. (D) Three types of standard nucleotide (Std-YY, Std-YH, and Std-HH) were prepared, which includes binary target sequences, corresponding to wild (Y) or mutant (H) variants, and annealing sites with the internal primers at each end. Tenfold serial dilusions of each standard were subjected to the second PCR. (E) The Invader assay for each standard was also performed in triplicate.
Mentions: Three nucleotide sequences, which also contained several degenerate sites, were designed for the Invader assay (Table 1). For the calibration of the proportion of mutant variants, three types of standard nucleotide were prepared: Std-YY, Std-YH, and Std-HH (Table 1). Each sequence includes binary target sequences, corresponding to wild (Y) or mutant (H) variants, and also includes annealing sites with the internal primers at each end. An additional four nucleotides were inserted between the binary target sequences to avoid interference. Dilution series of these standards were assayed in triplicate with each assay. For Invader assays, 384-well reaction plates were used with the ABI Prism 7900 HT sequence detection system (Life Technologies, Foster City, CA, USA). The plate was incubated isothermally at 63°C and fluorescence intensities were detected every 2 min for 40 min or until non-specific fluorescence [21] was detected. Each sample was tested in triplicate in the same plate. A flow diagram representing a method of nested-PCR followed by Invader assay is depicted in Fig 3.

Bottom Line: Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed.Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain.Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.

ABSTRACT
Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

No MeSH data available.


Related in: MedlinePlus