Single LDL apheresis improves serum remnant-like particle-cholesterol,C-reactive protein,and malondialdehyde-modified-low-density lipoprotein concentrations in Japanese hypercholesterolemic subjects

BACKGROUND: We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. METHODS: DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells. RESULTS: The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%. CONCLUSIONS: PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.     

K-ras point mutations in spontaneously occurring endometrial adenocarcinomas in the Donryu rat

The Donryu rat has been found to have a high incidence of spontaneous uterine endometrial carcinomas. Moreover the histologic findings, biological nature and pathogenesis of these rat tumors appear similar to those in humans. To determine if the incidence of H- and K-ras gene mutations in these rat tumors is similar to that in human endometrial cancers, we isolated DNA samples from 2 atypical hyperplasias, 5 simple or complex hyperplasia without atypia, 9 adenocarcinomas and 7 histologically normal tissues, amplified exons 1 and 2 of the H- and K-ras genes by PCR and hybridized the products with allele specific oligonucleotide probes. K-ras point mutations were observed in 1/2 of the atypical hyperplasia (codon 12: GGT-->GTT) and 3/9 of the carcinoma (codon 12: GGT-->GAT, GGT-->AGT, codon 61: CAA-->CAC), while they were not detected in 7 of the normal tissues and in 5 of the simple or complex hyperplasia without atypia. H-ras point mutations were not detected in any of these DNA samples. These frequencies in this rat model are similar to those in humans. The absence of K-ras mutations from simple and complex hyperplasia tissue samples suggests that these mutations are associated with cytological atypia. Our findings suggest that alterations in the K-ras gene may be one of the important initiating event in endometrial carcinogenesis in some of the Donryu rat, like the human.     

The effect of EDTA contaminated in sera on laboratory data

BACKGROUND: Two types of quantitative assay kits for K-ras mutations, PCR-preferential homoduplex formation assay (PHFA) and enriched PCR and enzyme-linked mini-sequence assay (ELMA), were recently developed. The K-ras mutations were analyzed using these assays. MATERIALS AND METHODS: DNA was extracted from the pancreatic juice which was obtained by endoscopy from 38 patients with pancreatic neoplasms (23 adenocarcinomas and 15 intraductal papillary neoplasms) and from 62 without it. RESULTS: The results of the two methods were mutually correlative. K-ras mutation was detected at high levels (mutant ras genes occupied >2% of all K-ras genes) in 25 of the 38 cases (66%) with pancreatic neoplasm. It was also detected at high levels in 9 of the 14 cases (64%) with pancreatic cyst. In contrast, the mutant gene was detected at a lower level (<2%) in other cases. CONCLUSIONS: Quantitative analysis of the mutant ras gene provided a useful tool for diagnosing the pancreatic carcinoma when the percentage of the mutant is high, especially when the types of mutation were either GAT or GTT.     

Analysis of point mutations at codon 12 of K-ras in human endometrial carcinoma and cervical adenocarcinoma by dot blot hybridization and polymerase chain reaction.

Samples of human endometrial carcinomas and cervical adenocarcinomas were screened for the presence of single site DNA mutations at codon 12 of the K-ras gene using dot blot hybridization of DNA amplified by the polymerase chain reaction (PCR). Of 21 cases of endometrial carcinoma, point mutations were observed in three cases (14.3%). Mutation from GGT to GTT was seen in one case, and mutation to GAT was observed in two cases. Of seven cases of cervical adenocarcinoma, point mutations were noted in two cases (28.6%). Mutation from GGT to GTT and double mutation to GAT and GCT were in one case each. However, no correlation was found between the presence of point mutation and age, clinical stage, or depth of muscular invasion. With respect to prognosis, of five patients with point mutation, one with cervical carcinoma died, and of 23 patients without mutation, one with endometrial carcinoma died.     

超声引导下细针穿刺胰腺腺癌基因诊断

目的：探讨超声引导下细针穿刺与基因检测相结合早期诊断胰腺癌的临床应用。方法：应用超声引导下细针穿刺技术对３８例可疑胰腺癌及其邻近器官占位性病变患者进行穿刺活检,同进采用聚合酶链反应－限制性酶切片段长度多态性技术,对所取得的标本ｃ－ｋｉ－ｒａｓ基因第１２密友子突变进行检测。应用于胰腺癌的诊断。结果：２２例胰腺癌中有２１例有ｃ－ｋｉ－ｒａｓ基因第１２密友子突变,多为ＧＧＴ变成ＧＡＴ、ＧＣＴ、ＧＴＴ,阳     

A relationship between K-ras gene mutations and some clinical and histologic variables in patients with primary colorectal carcinoma.

Mutations in the Kirsten ras 2 (K-ras) gene were described as early events in the process of colorectal carcinogenesis. The aim of this study was to find a possible relationship between the presence of K-ras mutation in samples of primary colorectal carcinomas and the clinico-pathological data of the investigated patients. Mutation in codon 12 of the K-ras gene was determined in 18 of 53 colorectal carcinomas (34%) in our group of patients. The presence of K-ras gene mutations was not related to gender, age of subject at diagnosis, staging or cancer location (p > 0.05). Sixteen of the 42 (38%) moderately differentiated carcinomas, and two of the eight (25%) well differentiated carcinomas contained K-ras mutation in codon 12, but none of the three poorly differentiated carcinomas contained the mutation. Moderately differentiated tumours contained an aspartate code GAT (in eight cases), a valine code GTT (in six cases), an alanine code GCT (in one case) and a serine code AGT (in one case) in codon 12. Well differentiated tumours contained only the valine code GTT (two cases). Our results show that the frequency of mutations in the K-ras gene in carcinomas in Central Europe is not different from the frequencies found in other parts of the world. The homogeneous incidence of K-ras mutation does not seem to be related to ethnic factors, dietary habits, or the composition of the diet.     

Melting temperature analysis as quantitative method for detection of point mutations.

Different methods have been devised to detect point mutations. Some are very sensitive, detecting mutations even in a background of normal tissue, but none provide information about the percentage of cells with mutant DNA. Here we describe an easy, fast and reliable method, melting temperature analysis, which not only detects point mutations but also provides quantitative information on the percentage of cells with mutant DNA. By this method we detected a G-A transition in codon 12 of the K-ras gene in DNA of subjects with colorectal cancer. The K-ras mutation was found in 9/10 bowel cancers and 8/10 normal adjacent samples. It was also detected in 4/7 stool samples from the same patients. In colorectal cancers, the proportion of K-ras mutant cells was variable: in two the mutant/wild-type DNA ratio was 30/70, in three 50/50, and in four 70/30. Melting temperature analysis was sensitive for the detection of point mutations in bowel cancer and also in apparently normal tissue, providing quantitative information about the percentage of cells with mutant DNA.     

K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification

BACKGROUND: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMS(TM) allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. METHODS: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. RESULTS: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04-100%. CONCLUSIONS: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.     

Ultrasensitive Detection of KRAS2 Mutations in Bile and Serum from Patients with Biliary Tract Carcinoma Using LigAmp Technology

Patients with biliary tract carcinoma have a poor prognosis. Early detection efforts are urgently needed to ameliorate the dismal prognosis for these patients. Mutations of the KRAS2 gene are one of the most common genetic aberrations in this cancer. In this study, we used LigAmp, an ultrasensitive technology for detecting point mutations, to analyze KRAS2 mutations in patients with a variety of neoplastic and non-neoplastic pancreatobiliary diseases. DNA was isolated from 64 samples, including 44 bile samples and 20 serum samples. Oligonucleotides specific for KRAS2 G35A (GAT, G12D), G35T (GTT, G12V), and G34A (AGT, G12S) mutations were used. KRAS2 mutations were detected in 14 of 16 (87.5%) neoplastic bile samples and in 9 of 28 (32.1%) non-neoplastic bile samples. However, the mutation levels were significantly lower in the non-neoplastic bile (median = 0.4%) compared with those in the neoplastic bile (median = 5.1%). KRAS2 mutations were also detected in 9 of 11 (81.8%) serum samples from patients with biliary tract carcinoma, which was further confirmed by cloning BstN1-refractory PCR products and DNA sequencing. However, KRAS2 mutations were not present in the sera from eight patients with benign pancreatobiliary diseases. These data demonstrate that KRAS2 mutations are detectable in both bile and serum using LigAmp. This technology has the potential for early biliary tract carcinoma detection and possibly for residual disease monitoring post-therapy.Biliary tract carcinoma arises within the bile duct system, and is anatomically divided into carcinoma of the gallbladder, as well as intrahepatic and extrahepatic cholangiocarcinoma. It carries a poor prognosis, with an overall 5 year-survival of less than 5%.¹ In the United States, intrahepatic cholangiocarcinoma is the second most common primary hepatic cancer, and cholangiocarcinoma as a whole accounts for >4500 cancer-related deaths each year. The dismal outcome of biliary tract carcinoma is largely because the majority of cancers are diagnosed at an unresectable advanced stage. Patients with early biliary tract carcinoma have a survival rate as high as 40% following a curative surgical resection,^2,3 whereas those diagnosed late have a survival rate of only 5%. Early detection therefore could have a significant impact on overall survival. Although recent progress has been made in various imaging modalities such as endoscopic retrograde cholangiopancreatography, endoscopic ultrasonography, and magnetic resonance imaging, diagnosis of biliary tract carcinoma at an early stage is still difficult in many cases.^4,5 In addition, it is technically challenging to perform endoscopic biopsy because of the presence of biliary strictures associated with small lesions. Although biliary tract carcinoma is derived from the bile duct epithelium, endobiliary brush cytology typically yields a low sensitivity of detection for malignancy.⁶ Furthermore, reactive biliary epithelial atypia associated with inflammatory biliary stricture mimics cancerous epithelium, which makes it problematic to diagnose biliary tract carcinoma cytologically and histologically. On the other hand, since this type of tumor tends to grow less coherently and the neoplastic cells are easily shed into bile, molecular analysis of tumor DNA in bile could be an important ancillary testing for definitive diagnosis of early biliary tract carcinoma.Patients with biliary tract carcinoma who harbor locally advanced or distant metastatic disease are not candidates for surgery, and receive either systemic chemotherapy or local irradiation. However, there are few avenues available to monitor the effectiveness of therapy, or to assess for minimal residual disease in the face of therapeutic response. To ensure that these patients receive effective treatment and avoid unnecessary toxicities, it is mandatory to have a sensitive and specific test to monitor the therapeutic response in vivo using a relatively non-invasive assay.Activation of oncogenes and inactivation of tumor suppressor genes have been identified as important contributors in the development of biliary tract carcinoma.^7,8,9 The incidence of KRAS2 mutations that have been reported in biliary tract carcinoma varies widely ranging from 0% to 100%, depending on what techniques have been used. KRAS2 mutations most commonly occur at codon 12, including GGT to AGT, GAT, and GTT.^9,10 Detection of KRAS2 mutations in bile has been explored as a potential diagnostic test by several groups. However, in these studies, although KRAS mutations were detected in the primary tumors of more than 80% cases, a much lower frequency of KRAS2 mutations was observed in bile from the same population,^6,11,12 which probably resulted from the presence of excessive wild-type DNA in the bile. An identical technological pitfall exists for detecting mutant DNA in serum. A sensitive and specific assay could improve the detection of mutant KRAS2 to a great extent.Recently, we developed an ultra-sensitive technology, LigAmp, for detection of single base mutations in the presence of large amounts of wild-type DNA.¹³ We have demonstrated that LigAmp is able to detect one mutant in the presence of ∼10,000 wild-type molecules or cells. In the present study, we used LigAmp to analyze KRAS2 mutations in the bile and serum from patients with a variety of non-neoplastic diseases and those with biliary tract carcinoma.     

同期性多灶性肺癌克隆来源的确定

应用分子生物学方法确定同期性多灶性肺癌的克隆来源 ,为肺癌的分期提供依据。方法 :对 2例肺部同期多灶性肺癌患者的每个病灶都用石蜡切片做显微解剖及蛋白酶K消化 ,提取基因组DNA ,先后两次PCR扩增K -ras第 1外显子和 p5 3外显子 5～ 9,用微型凝胶做非同位素性SSCP电泳 ,银染色后观察。对有变异电泳图型的样本用DNA序列测定去证实点突变。结果 :例 1的两个肺癌结节 ,仅 1个表现K -ras第 1外显子 12密码子突变 (GGT→TGT) ;例 2的 3个肺癌结节 ,1个表现K -ras第 1外显子 12密码子突变 (GGT→TGT) ,另 1个表现 p5 3第 5外显子 48密码子突变 (GTG→TTG) ,第 3个无突变发现。结论 :此 2例的多灶性肺癌均为不同的克隆来源。因此 ,提出应用非同位素PCR -SSCP分析 ,检测K -ras和p5 3点突变是鉴别多发性肺癌克隆起源的有效方法。     

焦磷酸测序法快速检测胰腺癌K-ras基因点突变

目的 建立基于焦磷酸测序技术的胰腺癌K-ras基因点突变的检测方法,并与Sanger测序法作一比较.方法 用焦磷酸测序法(Pyrosequencing)和Sanger测序法(Sanger sequencing)分别在10名正常胰腺组织、49例胰腺癌、11例慢性胰腺炎、18例胰腺良性囊肿、7例胰岛素癌、9例壶腹癌、7例胆管癌及7例十二指肠乳头癌石蜡包埋组织的DNA中检测K-rag基因12密码子点突变.结果 采用上述两种方法在所有正常胰腺组织、慢性胰腺炎、胰腺良性囊肿、胰岛素癌、壶腹癌、胆管癌及十二指肠乳头癌均未见K-ras基因点突变,而采用焦磷酸测序法发现胰腺癌石蜡包埋组织K-ras基因点突变率为71.4%(35/49),显著高于采用Sanger测序法发现胰腺癌石蜡包埋组织K-rag基因点突变率(61.2%,30/49).结论 焦磷酸测序法较Sanger测序法更为敏感,且焦磷酸测序法准确、快速、高通量,适合临床标本的批量测定.     

Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice

Approximately 15-30% of human non-small cell lung cancers (NSCLC) carry K-ras mutations, among which point mutations at codon 12 are the most common. This study characterizes the anti-tumor effect of an anti-K-ras ribozyme adenoviral vector (KRbz-ADV; replication-deficient, E1-deleted Ad5 backbone) against NSCLC lines that express the relevant mutation (K-ras codon 12 GGT --> GTT; H441 and H1725). KRbz-ADV significantly inhibited tumor cell growth (38-94% reduction by 3H-thymidine uptake) in a time- and dose-dependent manner, but produced minimal growth inhibition on normal epithelial cells, or NSCLC H1650 cells that lack the relevant mutation. The in vivo anti-tumorigenic effect of KRbz-ADV treatment was characterized with cell line xenografts in nu/nu mice. Pre-treatment with KRbz-ADV (10 or 20 p.f.u. per cell) completely abrogated subcutaneous engraftment of H441 (n = 13) or H1725 cells (n = 8), as compared with a 100% tumor take and progressive tumor growth in animals that received untreated tumor cells, or control vector (luciferase-adenovirus/Luc-ADV)-treated tumor cells. Pre-treatment with a mutant anti-K-ras ribozyme adenoviral vector (mutKRbz-ADV), which has the same specificity as KRbz but lacks ribozyme catalytic activity, did not produce an anti-tumorigenic effect. The in vivo effect of KRbz-ADV treatment was further examined by initiating injections (2 x 10(9) p.f.u.) at 7 days after tumor induction. Pre-existing tumor growth was reduced by 39% by a single intratumoral injection. Repeat injections (three or five KRbz-ADV-intratumoral injections at 2 x 10(9) p.f.u. every other day) resulted in complete tumor regression in five of seven mice. In contrast, single or multiple injections of control vector Luc-ADV did not significantly alter tumor xenograft outcome. Ribozyme expression was confirmed in H441 cells that demonstrated reduced growth after KRbz-ADV treatment. Reduced growth corresponded to significantly lowered levels of K-ras mRNA, as defined by RT-PCR (51% of untreated level, n = 3) and RNase protection assay (56% of untreated level, n = 4) analyses. Further, 37.5% of KRbz-ADV-treated cells underwent apoptosis, as compared with 11.7%, and 19.0% in untreated and Luc-ADV-treated cultures, respectively. A significantly higher proportion of KRbz-ADV-treated H441 cells (58.2%) underwent apoptosis when maintained under anchor-independent conditions that simulate in vivo tumorigenesis ('anoikis'). This is the first report that demonstrates that KRbz-ADV can effectively inhibit in vivo tumorigenesis, and produces regression of pre-existing human lung tumor xenografts having the relevant K-ras mutation.     

Linked linear amplification for simultaneous analysis of the two most common hemochromatosis mutations

BACKGROUND: Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations. METHODS: Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin-horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared. RESULTS: The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63. CONCLUSIONS: LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.     

ME-PCR for the identification of mutated K-ras in serum and bile of pancreatic cancer patients: an unsatisfactory technique for clinical applications

Our aim was to assess the clinical reliability of mutated K-ras detection in serum or bile for the diagnosis of pancreatic cancer using ME-PCR. DNA was extracted from 1 ml serum obtained from 29 patients with pancreatic cancer and 12 control subjects. ME-PCR was optimized using a mixture of normal DNA added with different amounts of mutated DNA. The analysis of sera obtained from the 29 patients and of bile obtained from 11 pancreatic cancer patients demonstrated the presence of mutated K-ras in two (6.9%) and four cases (36%). By contrast K-ras was not amplifiable in any of the 12 serum samples obtained from healthy controls. In conclusion the DNA obtained from pancreatic cancer patients' sera is suitable for K-ras amplification and for the identification of codon 12 point mutations. However ME-PCR alone has an unsatisfactory sensitivity for the detection of pancreatic cancer using serum DNA as starting template.     

急性髓性白血病N-ras基因突变的检测及临床意义

目的 研究急性髓性白血病（AML）患者中N-ras基因突变的检出率及其临床意义.方法 采用聚合酶链式反应（PCR）及序列分析方法检测117例初治AML患者N-ras基因第12、13和61位密码子的突变情况.结果 在117例AML患者中,发现N-ras基因突变9例,包括50例FAB分型M2中1例,35例M4中5例,16例M5中2例及6例M6中1例,而在2例M0、2例M1及6例M3中未发现N-ras基因突变,总检出率为7.7%.共检出3例第12密码子GGT→GAT突变,1例第13密码子GGT→GAT突变和5例第61密码子突变（包括3例CAA→CGA突变,1例CAA→AGA突变,1例CAA→AAA突变）.N-ras突变在正常核型中的检出率（4/30,13.3%）高于在异常核型中的检出率（1/19,5.3%）,但差异无统计学意义（P〉0.05）.结论 在AML患者中可以检测到N-ras基因突变,该突变可发生在N-ras基因的第61、12和13密码子,可能在AML发病机制中发挥一定作用.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.     

通用模板PCR技术快速检测乙肝病毒YMDD变异株

目的　探讨快速、安全地检测血清中对拉米夫定耐药的乙型肝炎病毒YMDD变异株。方法　用UT- PCR法结合Taqman荧光探针技术 ,针对HBVcodon 5 5 0位设计两条高度特异性引物Pi和Pv ,并设置保守区对照引物Pc ,在HBV耐药检测中使用了 3管阵列 ,经在PE70 0 0上进行PCR后 ,根据△Cti和△Ctv值 ,来判断是否发生了YVDD和YIDD突变。结果　检测 163例经拉米夫定治疗的病人血清 ,5 1例YMDD变异株阳性 ,其中随机选 15例样本进行DNA测序 ,结果完全相符。结论　本方法具有快速、准确、安全等特点 ,值得推广。     

蝎子PCR法与传统测序法检测K-ras基因突变的比较

目的建立一种简单、便捷、有效检测K-ras突变的蝎子PCR新方法.方法同时用蝎子PCR法和Sanger测序法对87例结直肠癌组织的K-ras基因进行热突变型G12D(GGT>GAT)进行检测,统计其符合率.结果87例结直肠癌组织样本中,蝎子PCR法和Sanger测序法检测到同样9例G12D突变,其符合率为100%.结论蝎子PCR法能有效检测到K-ras突变,且比Sanger测序法更为简便、快速,适合临床应用.     

Rapid semiquantitative real-time PCR for the detection of human cytomegalovirus UL97 mutations conferring ganciclovir resistance

BACKGROUND: The development of infections with ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) remains a serious problem in recipients of stem cell or organ transplants. Nearly all GCV-resistant clinical isolates have mutations in the viral UL97 gene. The rapid detection of GCV-resistant HCMV infections is necessary and the relative proportions of wild-type and mutant strains are predictive for the efficiency of antiviral therapy. To date, genotypical resistance screening has been limited to restriction fragment length polymorphism (RFLP) and sequencing analyses. Here, we present a comprehensive real-time PCR approach for the detection of most frequent mutations in the UL97 gene associated with GCV resistance. METHODS: The laboratory strains AD169 and Towne, different wild-type isolates and plasmids constructed by site-directed mutagenesis and overlap extension with specific point-mutations in the UL97 gene were analysed by LightCycler PCR and compared with UL97 RFLP and sequencing analyses. RESULTS: A new and comprehensive set of LightCycler PCRs was created using specific hybridization probes with melting-point analysis for the relevant codons 594, 595, 603 and 607. Different wild-type isolates and plasmids containing specific UL97 mutations conferring GCV resistance were investigated in the real-time PCR assay. Total processing time was 80 min per assay, whereas combinations of RFLP and sequencing needed at least 3-4 days. Proportions of co-existing wild-type and mutant strains in mixed viral populations can be obtained. CONCLUSIONS: We established a rapid real-time PCR approach for the detection of most frequent HCMV UL97 mutations associated with GCV resistance. Moreover, the method allows semiquantitative differentiation of the proportions of co-existing wild-type and mutant strains. This approach represents a new alternative for laborious RFLP analysis.     

Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique

ObjectivesThe objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing.Design and methodsWe developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods.ResultsThe RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing.ConclusionsCompared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of low-level KRAS mutations in the clinic.