RT-ARMS-qPCR定量测定线粒体耳聋患者mtDNA A1555G位点突变

目的 探讨RT-ARMS-qPCR(real time amplification refractory mutation system quantitativePCR)系统定量测定线粒体DNA(mitochondrial DNA,mtDNA)A1555G位点突变在线粒体耳聋发病机制研究中的价值.方法 以PeR扩增含mtDNA 1555位点的片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品;在引物3'端插入错配碱基AC建立RT-ARMS-qPCR系统,对含突变型和野生型mtDNA 1555位点片段的12例线粒体耳聋患者进行定量检测.结果 RT-ARMS-qPCR系统在检测1个含野生型mtDNA 1555的重组质粒DNA模板时,其批内变异系数(CV)为1.34%,批间CV为1.96%,线性范围为102-108拷贝/ul;突变型或野生型引物只特异扩增相对应的序列,特异性好;突变型/野生型mtDNA拷贝数及突变型所占的百分比与耳聋的严重程度相关(r=0.771,P=0.003),突变型mtDNA所占的比例越高,耳聋程度越严重.结论 RT-ARMS-qPCR系统适合于定量检测mtDNAA1555G点突变的线粒体DNA片段,结果特异、稳定、准确.线粒体耳聋的严重程度与含突变型和野生型mtDNA 1555位点的拷贝数比例有关.     

Determination of RhD zygosity: comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach

BACKGROUND: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. METHODS: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, DeltaCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. RESULTS: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the DeltaCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P: <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. CONCLUSION: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.     

Detection and quantification of minority HIV isolates harbouring the D30N mutation by real-time PCR amplification

OBJECTIVES: HIV drug resistance is a major concern as the emergence of resistant strains of virus results in failure of first-line therapies with an associated increase in the cost of subsequent regimens. Genotypic resistance is currently assessed by direct sequencing and cannot detect resistant species below 20%. Real-time PCR amplification was assessed for its ability to detect the signature mutation for nelfinavir, D30N. METHODS: A real-time PCR assay was optimized for detection of low levels of D30N and tested on in vitro-generated nelfinavir-resistant isolates as well as 10 clinical isolates (which were also characterized by sequencing). RESULTS: The sensitivity of the assay was 1% and quantification was possible as low as 4% of the total viral population. Furthermore, this methodology enabled quantification of the 30N mutation in two isolates shown to be negative by sequencing. CONCLUSIONS: Real-time PCR is a promising tool for the detection of minority species of HIV but further studies are required to determine the specificity of the assay in a larger and thus more diverse set of clinical isolates.     

Assessing heteroplasmic load in Leber's hereditary optic neuropathy mutation 3460G->A/MT-ND1 with a real-time PCR quantitative approach

To quantify the amount of the 3460G-->A/ND1 point mutation responsible for Leber's hereditary optic neuropathy, we developed a quantitative real-time polymerase chain reaction method based on the SYBR Green assay and a new approach using the TaqMan assay. Both methods were based on the amplification refractory mutation system, comparing the heteroplasmic load quantified by restriction fragment length polymorphism in 15 Leber's hereditary optic neuropathy family members, with the results obtained using quantitative real-time polymerase chain reaction methods. The comparative evaluation of mitochondrial DNA (mtDNA) heteroplasmy from blood samples showed significant correlation between restriction fragment length polymorphism analysis, real-time SYBR Green assay, and TaqMan assay. We validated the last method by measuring experimental samples composed by a known proportion of cloned plasmids containing either the wild-type or mutant sequence, giving a correlation coefficient of 0.999 (P < 0.0001). The real-time amplification refractory mutation system polymerase chain reaction by TaqMan assay provides a rapid, reliable, sensitive, reproducible, and one-step quantitative method to detect heteroplasmic mutant mtDNA. This method allows the quantitation of a broad range of mutational load (up to 100%, down to 0.01%) on the basis of in vitro calibration, thus rendering the TaqMan assay suitable for the diagnostic analysis of heteroplasmic load in mtDNA-related disorders.     

Identification of G6PD Mediterranean mutation by amplification refractory mutation system

Aim: To examine the plasma nitrate/nitrite (NOx—two end products of the nitric oxide metabolism) and endothelin (ET) concentrations, and response to acute adrenaline induced hypertension in diabetic rats. Materials and methods: Four groups of 4-month-old rats were used: control rats (C, n=10) rats received adrenaline (A, 40 μg/kg i.v., n=10), rats received streptozotocin (S, 50 mg/kg i.v., n=8), and rats received STZ and adrenaline (SA, n=9). The experiments were performed 4 weeks after the STZ administration. Plasma NOx, ET, glucose, and mean arterial blood pressure (MAP) were measured. Results: Plasma ET concentrations were significantly increased in diabetic rats (S and SA) in comparison with the controls and adrenaline-only administered rats. NOx concentrations in diabetic groups (S and SA) were significantly decreased in comparison with the controls. Acute adrenaline induced hypertension in diabetes leads to a significant decrease of NOx concentrations in comparison with the controls, adrenaline-only administered and STZ-only administered rats. There was no difference between the MAP in diabetic and control rats. Adrenaline injection caused a significant increase of MAP in A and SA groups. Plasma glucose concentrations in diabetic rats (S and SA) were significantly increased in comparison with the nondiabetic groups (C and A). There was a weak but significant correlation between the NOx and ET concentrations in the controls, which probably reveal the balance between these vasoactive factors. In A, S, and SA groups, no significant correlation between the NOx/ET was found. Conclusion: An impairment of the NOx and ET formation could be involved in the pathogenesis of diabetes mellitus and especially acute hypertension and diabetes. A lack of correlation between the NOx and ET probably indicated that in diabetes and acute hypertension, a primary mechanism of compensatory nitric oxide might be lost.     

Detection and monitoring of minimal residual disease by quantitative real-time PCR

BACKGROUND: The detection of malignant cells by quantitative real-time PCR has become state of the art for diagnosis, monitoring response to treatment and detection of minimal residual disease (MRD) in patients with leukemia or lymphoma. In order to be used in high-throughput analyses technical details have to be standardized to improve reproducibility and comparability of quantitative results obtained in different laboratories. METHODS: Molecular monitoring of disease activity during and after treatment based on the detection of malignant cells in circulation or bone marrow by quantitative real-time PCR will be helpful to develop individualized treatment strategies for every patient. CONCLUSIONS: The effectiveness of any kind of innovative treatment with specific antibodies, cellular immunotherapy or molecules designed for specific targets of tumor cells can be controlled at a very high level of sensitivity and accuracy. Based on quantitative results indicative for success or treatment failure, therapeutic changes upon the detection of progressive disease at the molecular level can be made even before symptoms or signs of clinical relapse occur. Hopefully, this will lead to higher cure rates and improved long-term survival.     

采用荧光定量PCR检测JAK2基因V617F突变

目的 建立荧光定量PCR检测JAK2基因V617F突变的方法,评估JAK2 V617F突变在诊断骨髓增殖性疾病和白血病中的临床意义.方法 选取71例慢性粒细胞性白血病(CML)、22例原发性血小板增多症(ET)、11例原发性骨髓纤维化(PMF)、9例真性红细胞增多症(PV)、7例嗜酸粒细胞增多症患者,分别采用荧光定量PCR、突变特异性扩增系统(ARMS)对JAK2 V617F突变进行检测,并采用基因测序对结果进行验证.将具有JAK2 V617F纯合子突变的人类红白血病细胞株(HEL)DNA作为阳性对照,比较荧光定量PCR和ARMS对JAK2基因V617F突变的检测敏感度.结果 采用荧光定量PCR检测,野生型的熔解温度(Tm)峰出现于(75.0±0.2)℃处,突变型的Tm峰出现于(76.6±0.2)℃处.JAK2基因V617F突变在PV、ET、PMF等骨髓增殖性疾病中的检出率分别为8例(88.9%)、12例(54.5%)、7例(63.6%),但在71例CML中只检出1例(1.4%).荧光定量PCR与ARMS的结果符合率为100%,结果经过测序证实.使用荧光定量PER法可在每106个正常自细胞中检出低至102个HEL细胞,而使用ARMS方法时要在每106个正常白细胞中HEL细胞达到104个时,方可检测出,前者比后者方法灵敏100倍.结论 成功地建立了荧光定量PCR检测JAK2基因V617F突变的方法,比ARMS更灵敏、简便,适合临床使用.JAK2基因V617F突变在骨髓增殖性疾病中有较高的检出率,可作为骨髓增殖性疾病特异性诊断指标.     

Detection and quantification of Shiga toxin-encoding genes in sheep faeces by real-time PCR

Sheep faeces may be an important source of Shiga toxin (Stx)-producing Escherichia coli. We have, therefore, established and evaluated a real-time 5'-nuclease PCR assay to quantify the stx(1) and stx(2) genes in sheep faeces. The detection limit of our assay for both stx(1) and stx(2) in spiked samples corresponded to 10(2)--10(3)CFU/g, which is lower than for other assays for measuring these genes in faecal samples. Quantification values for our assay ranged from 10(2) to 10(7)CFU/g faeces. The assay was evaluated on native, un-spiked faeces. All sheep tested (n=7) shed stx(1), and the quantitative results corresponded to the gene copies in 10(3)--10(4)CFU/g. The level of stx(2), however, was below the quantitative detection limit in all the samples analyzed. This quantitative stx(1) and stx(2) assay may be important in assessing whether sheep harbouring Shiga toxin-producing bacteria represent a potential hazard to human health.     

实时荧光定量PCR检测血浆结核分枝杆菌DNA的初步临床应用

目的 评价实时荧光定量PCR技术检测血浆（或血清）结核分枝杆菌（Mycobacterium tuberculosis,MTB）DNA的技术。方法 建立实时荧光定期量PCR方法测定血浆MTB DNA,分别检测临床确诊的55例结核病患者血清43份,血浆25份以及非结核肺部疾病患者血清18份,健康体检血清18份和健康体检者血浆11份的MTB DNA含量。结果 18例非结板肺疾病患者血清、18例健康体检者血清以及11例健康体检者血浆MTB DNA全部阴性。55例初诊结核患者中10例（18．2％）治疗前血浆（或血清）MTB DNA阳性。在痰涂片阴性的18例结核患者中,血浆（或血清）MTB DNA阳性检出率为27．8％（5／18）,其特异性达100％。结论 本研究证实了结核患者血浆（或血清）循环MTB DNA的存在,其定量检测对痰涂片阴性及无痰患者的结核病诊断有重要参考价值。     

Detection of C1236T,G2677T/A,and C3435T polymorphism of MDR1 by amplification refractory mutation system PCR

C1236T, G2677T/A, and C3435T polymorphism of the multidrug resistance (MDR1) gene have substantial impact on expression or activity of P‐glycoprotein (P‐gp). We developed new methods based on amplification refractory mutation system (ARMS) to detect these polymorphisms. Tetra‐primers amplification in a single tube was established to detect C1236T and C3435T polymorphism. For G2677T/A polymorphism, a two‐step allele‐specific amplification method was used. MDR1 genotypes of 177 Chinese subjects were determined by the methods we established. The methods we established were verified with gene sequencing. Gene frequencies of 1236C and 1236T were 37.8 and 62.2%, respectively; gene frequencies of 2677G, 2677T and 2677A were 44.1, 38.4 and 17.5%, respectively; the gene frequencies of 3435C and 3435T were 65.0 and 35.0%, respectively. The results were similar with other studies on Oriental subjects. The methods we established are simple, accurate, and economical, and can provide reliable approaches for determining MDR1 polymorphism. J. Clin. Lab. Anal. 23:110–116, 2009. © 2009 Wiley‐Liss, Inc.     

实时荧光定量PCR方法检测青藏高原喜马拉雅旱獭血样中布鲁氏菌DNA

  目的   应用实时荧光定量PCR（real-time PCR）方法检测青藏高原喜马拉雅旱獭血样中布鲁氏菌DNA。  方法   2019—2020年收集青海省青藏高原喜马拉雅旱獭全血,进行布鲁氏菌病血清学试验,包括虎红平板凝集试验（RBT）、试管凝集试验（SAT）微量法、胶体金免疫试验（GICA）,阳性标本进行real-time PCR检测。 分析real-time PCR方法的灵敏度、特异度等指标,评价其结果的准确性。  结果   共收集全血1 466份,RBT检出阳性64 份,GICA检出阳性28份,SAT检出阳性18份。64 份RBT阳性标本进行real-time PCR检测,其中56份标本及阳性对照菌株有特异的荧光扩增曲线,8份标本及阴性对照无特异的扩增曲线。 real-time PCR与SAT、GICA比较灵敏度为100%,与RBT比较特异度为99.93%,Kappa值为0.81,高度一致。  结论   与传统方法相比,real-time PCR方法具有快速、特异等优点,可以用于青藏高原喜马拉雅旱獭样本检测。     

IgH重排的实时定量PCR检测及反应参数研究

为了探讨以通用引物应用PCR技术扩增克隆性免疫球蛋白重链基因（IgH）重排的最佳实验条件及SYBR Green Ⅰ实时定量PCR检测该基因的可行性，以通用引物对克隆性IgH重排进行扩增，对影响PCR扩增的退火温度、引物浓度、Taq酶用量、dNTP浓度、镁离子浓度、循环次数等实验因素进行了系统研究，找出最佳反应参数。并以通用引物进行了SYBR greenⅠ实时定量PCR对IgH重排基因的检测，测定了该法检测IgH重排基因的敏感性。结果表明：最佳退火温度为60℃，最佳引物浓度为0．8μmol／L，0．5U的Taq酶量效果满意，最适dNTP浓度为100μmol／L，最适镁离子浓度为3．0mmol／L，最佳循环次数为40次。SYBR GreenⅠ实时定量PCR可以实现对克隆性IgH重排的扩增和荧光信号的检测分析，其对IgH重排基因检测的敏感性为10^4／ml。结论：确定了应用PCR技术检测克隆性IgH重排的最适反应条件，实现了用通用引物对克隆性IgH重排稳定、特异的扩增，初步实现了以通用引物和应用SYBR Green Ⅰ实时定量PCR对IgH重排的检测。