Refractory Mutation System Quantitative PCR Research Articles

Detection of BRAFV600E mutation of thyroid cancer in circulating tumor DNA by an electrochemical-enrichment assisted ARMS-qPCR assay

Qualitative and quantitative analysis of carcinogenic mutations in circulating tumor DNA (ctDNA) plays a unique role in the early screening and precise diagnosis of cancer. Herein, we introduced an electrochemical-enrichment assisted amplification refractory mutation system-quantitative real-time PCR (EC-ARMS-qPCR) assay to detect the BRAFV600E carcinogenic mutation of thyroid cancer in plasma samples. Cell-free DNA (cfDNA) were firstly enriched by an electric field-driven adsorption and desorption process, and then ctDNA in cfDNA were analyzed by a designed allele-specific qPCR assay. The EC-ARMS-qPCR assay provides good sensitivity and specificity for the identification of single nucleotide polymorphisms (SNPs) of BRAFV600E mutant ctDNA in a complex system, which can detect as low as 2 BRAFV600E mutant copies in Tris buffer and 25 BRAFV600E mutant copies in Tris buffer containing 50% plasma with a allele frequency of 0.01% (i.e., mutant-to-wild-type ratio of 1: 9999). The assay was assessed in plasma samples of patients with papillary thyroid cancer (PTC) and benign thyroid nodule (TN). The EC-ARMS-qPCR assay detected 40.74% and 10.00% BRAFV600E mutation ctDNA in plasma samples of 54 PTC patients and 20 TN patients, respectively. The overall concordance between BRAFV600E mutant ctDNA by EC-ARMS-qPCR assay and BRAFV600E mutant of matched pathological tissue genes by fine needle aspiration biopsy (FNAB) is 73.08%, suggesting that the EC-ARMS-qPCR assay is appropriate for detecting carcinogenic mutations in liquid biopsy samples.

Effect of ARMS-qPCR on detection sensitivity of earlier diagnosis of papillary thyroid cancers with worse prognosis determined by BRAF V600E and TERT promoter mutation coexisting.

6588 Background: Co-existing of BRAF V600E and TERT promoter C228T/C250T mutation has been extensively related to prognosis in thyroid cancer. Our study aimed to establish a more sensitive method for mutation detection and explore the correlation more in-depth. Methods: BRAF and TERT promoter mutation status of 250 papillary thyroid cancer was detected by both Amplification Refractory Mutation System quantitative PCR (ARMS-qPCR) and Sanger sequencing to compare the sensitivity. The associations between the mutation status and the clinicopathological features were analyzed. Results: ARMS-qPCR displayed higher sensitivity than Sanger ( BRAF V600E: 75.2% vs. 52.4%, p< 0.001; TERT promoter C228T/C250T: 12.0% vs. 3.6%, p= 0.001; Co-mutation (9.6% vs. 3.2%, p= 0.005). Both methods indicated that patients with BRAF V600E and TERT promoter co-mutation were higher in age at diagnosis (ARMS-qPCR: 51.0 ± 14.2 vs. 40.2 ± 12.6, p <0.001; Sanger: 64.3 ± 7.1 vs. 40.5 ± 12.6, p <0.001), and the recurrence rate (16.7% vs. 3.1%, p= 0.014; 50.0% vs. 2.9%, p< 0.001), besides, the co-mutation group were related to more advanced TNM stage ( p< 0.001; p< 0.001) and higher MACIS score (5.1 ± 1.5 vs. 4.2 ± 0.7, p= 0.006; 6.6 ± 1.1 vs. 4.2 ± 0.8, p< 0.001). In addition, compared with the co-mutation results of Sanger, it seems that ARMS-qPCR has identified an earlier stage of group, which were younger (43.3 ± 10.1 vs. 66.4 ± 6.1, p< 0.001), and with smaller tumor (1.8 ± 1.5 vs. 4.0 ± 1.3, p= 0.002), as well as lower recurrence rate ( 0.0% vs. 50%, p= 0.007). Besides, the newly identified group were lower in MACIS score (4.2 ± 0.8 vs. 6.9 ± 0.7, p= 0.002) and with lower TNM stage ( p= 0.001). Conclusions: Patients with BRAF V600E and TERT promoter C228T/C250T co-mutation have a worse prognosis. Using ARMS-qPCR, the more sensitive method could identify earlier stages of patients with a potentially worse prognosis. [Table: see text]

Successful reproduction of adenoviral gizzard erosion in 20-week-old SPF layer-type chickens and efficacious prophylaxis due to live vaccination with an apathogenic fowl adenovirus serotype 1 strain (CELO)

Recently, outbreaks of adenoviral gizzard erosion (AGE) have been documented in pullets and layers housed free range and in enriched cage systems characterized by increased mortality and a negative impact on egg production. In the present study the pathogenicity of a fowl adenovirus serotype 1 (FAdV-1) field strain as well as the aetiological role of a FAdV-8a strain, both isolated from AGE affected pullets, were investigated in vivo in 20-week-old specific-pathogen-free (SPF) layer-type chickens. Furthermore, the efficacy of a single (week 17) and double (week 14 and 17) application of a live vaccine consisting of an apathogenic FAdV-1 (CELO strain) against challenge with virulent FAdV-1 was investigated.For the first time, AGE was successfully reproduced in adult birds after oral infection of 20-week-old SPF birds with a virulent FAdV-1 field isolate, characterized by pathological changes of the gizzard from 7 days post challenge onwards. In addition, a negative impact of the FAdV-1 infection on the development of the reproductive tract was observed. Thus, confirming the pathogenicity and aetiological role of FAdV-1 in the development of AGE and economic losses due to AGE in layers. In contrast, no pathological changes were observed in birds infected with FAdV-8a.Independent of a single or double application of the live FAdV-1 vaccine strain CELO, no gross pathological changes were observed in gizzards post challenge with the virulent FAdV-1, indicating that complete protection of layers against horizontal induction of AGE was achieved. Nonetheless, virulent FAdV-1 was detected in cloacal swabs and gizzards in both vaccinated groups post challenge determined by the application of an amplification refractory mutation system quantitative PCR used to differentiate between vaccine and challenge strains.

JCES01.22 Comparison of Four Leading Technologies for Detecting EGFR Mutations in Circulating Tumor DNA from Patients with Non-Small Cell Lung Carcinoma

This study aimed to assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations including L858R, E19-dels, T790M, and G719X from circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC). Plasma samples were collected from 20 patients with NSCLC including detailed clinical information along with data regarding treatment response. ctDNA was extracted from 10 mL plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Extracted ctDNA was analyzed using two real time-amplification refractory mutation system-quantitative PCR platforms (cobas® EGFR Mutation Test: cobas; and AmoyDx® EGFR 29 Mutations Detection Kit: ADx), one digital platform (Droplet DigitalTM PCR, ddPCR: Bio-rad), and one next-generation sequencing platform (firefly NGS: Accuragen). If a positive result was obtained from any one of the four platforms, the sample was categorized as positive. We identified 15 EGFR mutations in 20 patients with NSCLC using the four platforms, for which 7, 11, 10, and 12 mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. Among the 15 EGFR mutations, six and seven EGFR alterations demonstrated an allele frequency of more or less than 1% (group A or B, respectively), and two exhibited unknown allele frequency. In group A, 5, 5, 5, and 6 EGFR mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. The positive coincidence rate of any two platforms ranged from 66.7% to 100% and the kappa value varied from 0.787 to 1.000 in group A. In group B, 1, 5, 5, and 6 EGFR mutations were detected and the positive coincidence rate of any two platforms ranged from 16.7% to 100% and the kappa value varied from 0.270 to 1.000. The output of cobas, ddPCR, and firefly NGS were highly correlated, whereas ADx displayed weak concordance with these three platforms in group B. In addition, we identified 75 wild-type loci when EGFR alleles identified as negative by one or more platforms were considered as negative. ADx, cobas, ddPCR, and firefly NGS uncovered 73, 69, 70, and 68 EGFR wild-type loci, respectively. The concordance and negative coincidence rates between any two platforms were over 90%. The detection rate and concordance were probably affected by the abundance of EGFR mutations and the sensitivity of different platforms. Three platforms, including cobas, ddPCR, and firefly NGS, exhibited higher positive coincidence and detection rates when the allele frequency was lower than 1%.

P3.02b-011 Comparison of Four Leading Technologies for Detecting EGFR Mutations in Circulating Tumor DNA from Patients with Non-Small Cell Lung Carcinoma: Topic: EGFR Biomarkers

This study aimed to assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations including L858R, E19-dels, T790M, and G719X from circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC). Plasma samples were collected from 20 patients with NSCLC including detailed clinical information along with data regarding treatment response. ctDNA was extracted from 10 mL plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Extracted ctDNA was analyzed using two real time-amplification refractory mutation system-quantitative PCR platforms (cobas® EGFR Mutation Test: cobas; and AmoyDx® EGFR 29 Mutations Detection Kit: ADx), one digital platform (Droplet DigitalTM PCR, ddPCR: Bio-rad), and one next-generation sequencing platform (firefly NGS: Accuragen). If a positive result was obtained from any one of the four platforms, the sample was categorized as positive. We identified 15 EGFR mutations in 20 patients with NSCLC using the four platforms, for which 7, 11, 10, and 12 mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. Among the 15 EGFR mutations, six and seven EGFR alterations demonstrated an allele frequency of more or less than 1% (group A or B, respectively), and two exhibited unknown allele frequency. In group A, 5, 5, 5, and 6 EGFR mutations were detected by ADx ,cobas, ddPCR, and firefly NGS, respectively. The positive coincidence rate of any two platforms ranged from 66.7% to 100% and the kappa value varied from 0.787 to 1.000 in group A. In group B, 1, 5, 5, and 6 EGFR mutations were detected and the positive coincidence rate of any two platforms ranged from 16.7% to 100% and the kappa value varied from 0.270 to 1.000. The output of cobas, ddPCR, and firefly NGS were highly correlated, whereas ADx displayed weak concordance with these three platforms in group B. In addition, we identified 75 wild-type loci when EGFR alleles identified as negative by one or more platforms were considered as negative. ADx, cobas, ddPCR, and firefly NGS uncovered 73, 69, 70, and 68 EGFR wild-type loci, respectively. The concordance and negative coincidence rates between any two platforms were over 90%. The detection rate and concordance were probably affected by the abundance of EGFR mutations and the sensitivity of different platforms. Three platforms, including cobas, ddPCR, and firefly NGS, exhibited higher positive coincidence and detection rates when the allele frequency was lower than 1%.

Genetic Variation of Bordetella pertussis in Austria.

In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.

Putative regulation mechanism for the MSTN gene by a CpG island generated by the SINE marker Ins227bp.

BackgroundA single nucleotide polymorphism (SNP) in the first intron of the myostatin gene (MSTN) is associated with aptness of elite Thoroughbreds to race over sprint, middle or long distances. This intronic marker (g.66493737 T ≻ C), a short interspersed nuclear element (SINE) of 227 bp (Ins227bp) insertion polymorphism in the MSTN promoter, and the adjacent SNP BIEC2-417495 have not been studied for their association with racing aptness of the average Thoroughbreds raced in countries with lower status of the racing industry. This study investigated these markers regarding their prevalence and association with performance in common race horses. Markers were genotyped by amplification refractory mutation system-quantitative PCR (ARMS-qPCR) or amplicon melting. Furthermore, we asked whether the Ins227bp marker might theoretically regulate the expression of myostatin by generating a novel target for DNA methylation or by changing binding sites for transcription factors. Putative sites for DNA methylation or binding of transcription factors were predicted by MethPrimer and by the softwares JASPAR, MatInspector and UniPROBE, respectively.ResultsPairwise linkage disequilibrium between g.66493737 T ≻ C and Ins227bp was high (r2 = 0.93). A lower linkage was determined for g.66493737 T ≻ C and BIEC2-417495 (r2 = 0.69) as well as for BIEC2-417495 and Ins227bp (r2 = 0.76). The estimated frequencies for the presence of Ins227bp (I) indel and the C alleles at g.66493737 T ≻ C and BIEC2-417495 were 0.46, 0.47 and 0.43, respectively. Heterozygotes represented the most abundant genotype at each locus. The best racing distance (BRD) was significantly different between the homozygotes of each SNP (p = 0.01 to 0.03). C allele homozygotes at BIEC2-417495 or g.66493737 T ≻ C, as well as Ins227bp homozygotes earned most money on a mean distance ranging from 1211 to 1230 m. Heterozygotes earned most money on races over 1690 to 1709 m. The BRD for the T/T carriers at both SNP loci and for the SINE-free genotype was 1812 to 1854 m. Other performance parameters were not significantly different between the genotypes, except of the relative success score (RSS). The RSS was significantly slightly better on a distance of ≤1300 m for all carriers of the C allele and the Ins227bp compared to homozygous T genotypes and SINE-negative horses (p = 0.037 to 0.046). For distances of more than 1300 m the RSS was not significantly different between genotypes.In silico assessment indicated that the Ins227bp promoter insertion might have generated a CpG island and a few novel putative binding sites for transcription factors.ConclusionsAll three target polymorphisms (Ins227bp, g.66493737 T ≻ C, BIEC2-417495) are suitable markers to assess the ability of non-elite Thoroughbreds to race at short or longer distances. The CpG island generated by Ins227bp may cause training-induced silencing of MSTN expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0428-3) contains supplementary material, which is available to authorized users.

Comparison of Genetic and Epigenetic Alterations of Primary Tumors and Matched Plasma Samples in Patients with Colorectal Cancer

BackgroundAlthough recent advances in circulating DNA analysis allow the prediction of tumor genomes by noninvasive means, some challenges remain, which limit the widespread introduction of cfDNA in cancer diagnostics. We analyzed the status of the two best characterized colorectal cancer (CRC) genetic and epigenetic alterations in a cohort of CRC patients, and then compared the degree to which the two patterns move from tissue to plasma in order to improve our understanding of biology modulating the concordance between tissues and plasma methylation and mutation profiles.MethodsPlasma and tumor tissues were collected from 85 patients (69±14 years, 56 males). KRAS and SEPT9 status was assessed by allele refractory mutation system quantitative PCR and quantitative methylation-specific PCR, respectively. Six of the most common point mutations at codon 12 and 13 were investigated for KRAS analysis.Results KRAS mutations and SEPT9 promoter methylation were present in 34% (29/85) and in 82% (70/85) of primary tumor tissue samples. Both genetic and epigenetic analyses of cfDNA revealed a high overall concordance and specificity compared with tumor-tissue analyses. Patients presenting with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses. The median methylation rates in tumour tissues and plasma samples were 64.5% (12.2–99.8%) and 14.5% (0–45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.8–86.3%) in tissues and 2.9% (0–17.3) in plasma samples. The plasma/tissue (p/t) ratio of SEPT9 methylation rate was significantly higher than the p/t ratio of KRAS mutation load, especially in early stage cancers (p=0.0108).ConclusionThe results of this study show a discrepant rate of epigenetic vs. genetic alterations moving from tissue to plasma. Many factors could affect mutation cfDNA analysis, including both presence of tumor clonal heterogeneity and strict compartmentalization of KRAS mutation profile. The present study highlights the importance of considering the nature of the alteration when analyzing tumor-derived cfDNA.

Clinical significance of periodic detection of hepatitis B virus YVDD mutation by ultrasensitive real-time amplification refractory mutation system quantitative PCR during lamivudine treatment in patients with chronic hepatitis B.

Monitoring hepatitis B virus (HBV) mutants periodically during nucleoside analogue treatment is of great clinical significance, particularly in persistently HBV DNA-positive patients. However, few studies have investigated the dynamic changes of HBV YMDD (Tyr-Met-Asp-Asp) and YVDD (Tyr-Val-Asp-Asp) populations in chronic hepatitis B (CHB) patients whilst undergoing lamivudine (LMV) treatment. In this study, we sought to investigate the dynamic changes of HBV YMDD and YVDD variants by ultrasensitive real-time amplification refractory mutation system quantitative PCR (RT-ARMS-qPCR) and evaluate its significance for changes in the treatment of CHB patients. RT-ARMS-qPCR was established and evaluated with standard recombinant plasmids. Fifteen CHB patients receiving LMV (100 mg daily) were consecutively recruited and followed up for 60 weeks. Serum samples were obtained from each patient at baseline and every 12 weeks. The total HBV DNA, HBV YMDD DNA and YVDD DNA levels were measured using RT-ARMS-qPCR at all given time points after treatment. Routine liver biochemistry parameters, including aspartate aminotransferase and alanine aminotransferase, were also measured every 12 weeks. The linear range of the assay was between 1×10(12) and 1×10(5) copies ml(-1). The low detection limit was 1×10(4) copies ml(-1). After 60 weeks of LMV treatment, nine patients experienced virological breakthrough. The YVDD variant could be detected 12-48 weeks before virological breakthrough. The YVDD variant was detected as the predominant population (range 69.4-100 %) in patients by the time virological breakthrough appeared. We concluded that RT-ARMS-qPCR was sensitive for the detection and quantification of low levels of HBV mutation. Periodic detection of HBV YM(V)DD every 12 weeks during LMV treatment is helpful for therapeutic decision making.

Infection with an apathogenic fowl adenovirus serotype-1 strain (CELO) prevents adenoviral gizzard erosion in broilers

Gizzard erosion in broilers due to an infection with virulent fowl adenovirus serotype 1 (FAdV-1) is an emerging disease. Although experimental studies were performed, a possible prevention strategy was not reported so far. The present study was set up to determine (i) a possible influence of birds’ age at time of inoculation on the pathogenicity of a European FAdV-1 field strain (PA7127), (ii) the virulence of a apathogenic FAdV-1 strain (CELO), and (iii) its capability to protect SPF broilers from adenoviral gizzard erosion caused by the field virus.Oral infection of birds with PA7127 at 1-, 10- and 21-days of life, resulted in reduced weight gain compared to non-infected birds, with significance for birds infected at day-old. Independent of the birds’ age at time of inoculation, clinical signs appearing approximately one week after challenge coincided with gizzard lesions.Birds infected exclusively with CELO at the first day of life did not show any clinical signs or pathological changes in the gizzard, confirming the apathogenicity of this European FAdV-1. A similar result was obtained for birds orally infected at the first day of life with CELO and challenged three weeks later with the pathogenic PA7127 strain. Therefore, complete protection of adenoviral gizzard erosion in broilers by vaccination of day-old birds could be demonstrated for the first time, although virus excretion was detected post challenge. Establishment of an amplification refractory mutation system quantitative PCR (ARMS-qPCR) facilitated the identification of the FAdV-1 strain and presence of challenges virus was confirmed in one sample.

Quantification of Mitochondrial DNA with the A1555G Mutation in Deaf Patients Using Real-Time Amplification Refractory Mutation System-Quantitative PCR.

The objective of this study was to develop a real-time amplification refractory mutation system-quantitative PCR (RT-ARMS-qPCR) assay for quantitation of mitochondrial DNA (mtDNA) with a mutated 1555 site and to explore the relationship between the degree of mtDNA1555 mutation and the clinical phenotype of mitochondrial deafness. An amplified mtDNA fragment containing the 1555 site was ligated into a vector to construct a plasmid DNA standard. An RT-ARMS-qPCR system was then used to measure the mtDNA copy number containing wild-type and mutant sequences in a cohort of 96 deaf patients. The variation coefficient in the cohort was 1.34%; the interassay variation coefficient was 1.96%; and the linear range was 10(2) to 10(8) copies/mul for detecting a recombinant, wild-type plasmid. The primers amplified only the intended sequences. Mutation copy number correlated with the degree of deafness in sporadic cases with heteroplasmic mutations of mtDNA A1555G (r = 0.771, P = 0.003), but not in sporadic cases with homoplasmic mutations (r = 0.001, P = 0.997). Furthermore, the copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G in familial cases correlated with degree of deafness (r = 0.341, P = 0.022 and r = 0.85, P = 0.015, respectively). The RT-ARMS-qPCR system was therefore suitable for determining the copy number of mtDNA fragments containing the A1555G mutation, and mtDNA A1555G copy number correlates with severity of hearing loss.

Heterogeneous patterns of tissue injury in NARP syndrome

Point mutations at m.8993T>C and m.8993T>G of the mtDNA ATPase 6 gene cause the neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome, a mitochondrial disorder characterized by retinal, central and peripheral neurodegeneration. We performed detailed neurological, neuropsychological and ophthalmological phenotyping of a mother and four daughters with NARP syndrome from the mtDNA m.8993T>C ATPase 6 mutation, including 3-T brain MRI, spectral domain optical coherence tomography (SD-OCT), adaptive optics scanning laser ophthalmoscopy (AOSLO), electromyography and nerve conduction studies (EMG-NCS) and formal neuropsychological testing. The degree of mutant heteroplasmy for the m.8993T>C mutation was evaluated by real-time allele refractory mutation system quantitative PCR of mtDNA from hair bulbs (ectoderm) and blood leukocytes (mesoderm). There were marked phenotypic differences between family members, even between individuals with the greatest degrees of ectodermal and mesodermal heteroplasmy. 3-T MRI revealed cerebellar atrophy and cystic and cavitary T2 hyperintensities in the basal ganglia. SD-OCT demonstrated similarly heterogeneous areas of neuronal and axonal loss in inner and outer retinal layers. AOSLO showed increased cone spacing due to photoreceptor loss. EMG-NCS revealed varying degrees of length-dependent sensorimotor axonal polyneuropathy. On formal neuropsychological testing, there were varying deficits in processing speed, visual–spatial functioning and verbal fluency and high rates of severe depression. Many of these cognitive deficits likely localize to cerebellar and/or basal ganglia dysfunction. High-resolution retinal and brain imaging in NARP syndrome revealed analogous patterns of tissue injury characterized by heterogeneous areas of neuronal loss.Electronic supplementary materialThe online version of this article (doi:10.1007/s00415-010-5775-1) contains supplementary material, which is available to authorized users.

Mitochondrial DNA A1555G mutation of seven families with nonsyndromic hearing loss

To study mitochondrial DNA (mtDNA) A1555G mutation in seven families with nonsyndromic hearing loss (NSHL). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real time-amplification refractory mutation system-quantitative PCR (ARMS-qPCR) were applied to detect mtDNA A1555G mutation in seven NSHL families. Related clinical data were also collected and analyzed. The mtDNA A1555G mutation was detected in members from the maternal side, including heteroplasmy and homozygosis, others were negative for this mutation. The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G correlated well with the degree of deafness (R = 0.341, P = 0.022 and R = 0.85, P = 0.015, respectively). The mutation rate of the mtDNA A1555G is high in the NSHL patients, the mutation type include heteroplasmy and homozygosis. There is significant correlation between the mtDNA A1555G copy number and the severity of hearing loss.

Quantitation of mitochondrial DNA A1555G mutation by real time amplification refractory mutation system quantitative PCR

Objective To develop a real time amplification refractory system(RT-ARMS-qPCR) quantitative PCR method with SYBR Green I to assess the mtDNA A1555G mutation. Methods A specific fragment flanking mtDNA 1555 site was amplified with PCR and ligated into a pGEM Easy T vector. Serial dilutions of the plasmid DNA were quantified the actual copy numbers were assessed using RF-ARMS-qPCR with SYBR Green I. RF-ARMS-qPCR was established with mismatched base pairs at 3' in the primer todetect the copy number of mtDNA containing wild or mutant mtDNA. The specificity of amplified products was checked by melting curve analysis. Results The intra- and interassay variation was 1.34% and 1.96%, respectively when the assay was used to detect 1 copy/ul recombinant template of plasmid. Thequantitative standard curve showed that the assay had good linear correlation from 102 copies/ul to108 copies/ul. This assay could be served for the quantification of other samples. There was significantcorrelation between frequency of mutant mtDNA and phenotype (r=0.771, P = 0.003) in hearing lossgroup. Conclusions The established assay can be used to detect quantitatively mtDNA A1555G mutation byRF-ARMS-qPCR. This assay is specific, stable and accurate. There is significant correlation betweenquantification of mtDNA AI555G and the severity of hearing loss. Key words: Hearing impaired persons; DNA, mitochondrial; Point mutation

Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach.

The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation. To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences. A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR-restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations. The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.