代表性差异分析和消减杂交差异显示分析方法在克隆差异基因方面的灵敏度比较

 目的　建立目的基因差异信息标量化的配对研究体系,比较经典的代表性差异分析（RDA）方法和消减杂交差异显示分析（SHDD）方法在克隆差异基因方面的灵敏度。方法　以人类乳头瘤病毒（HPV）DNA为模板行PCR扩增获得376 bp和869 bp两个片段,作为差异目的基因掺入人血基因组DNA,获得5组目的基因拷贝数呈梯度差异的配对研究体系,比较RDA 和SHDD方法的检测灵敏度。结果　应用RDA方法可克隆得到上调6倍的376 bp片段和上调8倍的869 bp的HPV片段,而应用SHDD方法可克隆到上调4倍的两种差异片段。结论　SHDD方法由于采用了两样本差异产物间的双向均衡消减杂交,避免了经典RDA方法不均衡消减杂交导致的差异信息的丢失,在克隆较弱的差异信息,尤其是较长差异信息方面,显著优于经典RDA方法。     

Clinical evaluation of a novel commercial multiplex‐based PCR diagnostic test for differential diagnosis of dermatomycoses

Dermatomycoses are very common worldwide with increasing prevalence. An accurate and rapid detection of fungi is most important for the choice of antimycotics and the success of treatment. The aim of this study was to evaluate a new commercial multiplex‐based PCR which allows the detection and differentiation of the most relevant human pathogen fungi causing dermatomycoses in Europe. The accuracy and reproducibility of this application were verified in a clinical performance assessment in comparison to direct microscopy and culture using DNA isolates from 253 clinical samples. Sensitivity, specificity, positive predictive value and negative predictive value of 87.3%, 94.3%, 87.3% and 94.3%, respectively, were calculated for dermatophytes when confirmed by direct microscopy, culture or both. The corresponding values for Candida spp. were 62.7%, 93.5%, 77.8%, and 87.4%, respectively. Furthermore, in comparison to culture, the multiplex PCR was able to detect additional 38 Trichophytum rubrum and 12 Trichophytum interdigitale infections. These results were confirmed by independent PCR analysis. From DNA isolation to diagnosis the multiparameter diagnostic kit gives rise to a 1‐day workflow, enables fast clarification of disease aetiology and, thus, contributes to specific therapy selection. The latter is particularly important in light of growing resistance to antimycotics.     

Mutational characteristics of young and elderly gastric cancer: a comparative study

BackgroundYoung gastric cancer (YGC) has been indicated as having a worse prognosis than in elderly gastric cancer (EGC). It has been reported that YGC and EGC patients show different genomic profiles; however, there has been no comparative study conducted to reveal their mutational characteristics.MethodsFirstly, we divided and analyzed the mutational landscape and 50 cancer-related genes characters of YGC (n=18) and EGC (n=18) patients from The Cancer Genome Atlas-Stomach Adenocarcinoma (TCGA-STAD). A total of 8 gastric cancer samples including 4 YGC and 4 EGC patients were collected to detect 50 cancer-related genes by multiplex polymerase chain reaction (PCR) next generation sequencing. The R/maftools package was used to describe the mutational characteristics.ResultsOur results showed that the EGC group harbored more mutations than the YGC group. In 50 cancer-related genes in our cohort, the YGC group tended to be different from the EGC group using multiplex PCR next generation sequencing. In the YGC group, candidate mutations were identified within the following genes: IDH2, PDGFRA, KRAS, FLT3, FGFR2, and FGFR3. The YGC group showed less tumor mutational burden (TMB) level then EGC.ConclusionsThe YGC group tended to be more sensitive to molecularly targeted therapy because of it having more somatic mutations in 50 cancer-related genes using targeted next-generation sequencing.     

Thymidylate synthase genotyping is more predictive for therapy response than immunohistochemistry in patients with colon cancer

Thymidylate synthase (TS) is a potentially valuable marker for therapy response since it is the molecular target of 5-fluorouracil (5-FU). TS can be analyzed at the DNA (gene polymorphisms and amplification) and protein level (immunohistochemistry). This study investigated the predictive role of TS at the DNA and protein levels in patients with N(+) colon cancer (n = 38). Tumor and normal tissues were genotyped using PCR for variable number of tandem repeats (VNTR), a single nucleotide polymorphism (SNP) in the 3R allele and a 6 bp deletion (1494del6) in the TS gene. Tumor tissues were additionally analyzed for loss of heterozygosity (VNTR polymorphism). A newly developed real time PCR assay was used to detect the presence of TS gene amplifications in tumor tissues. VNTR analysis in normal tissue was significantly associated with distant tumor recurrence (8% for 2R/2R vs. 52% for patients carrying a 3R allele, p = 0.038) and cancer-specific survival (p = 0.021). IHC was not found to be significantly associated with patients' outcome. No correlations between TS gene polymorphisms and IHC were found. However, TS gene amplification was correlated with a strong IHC staining intensity. In conclusion, this study indicates that DNA based analysis is more predictive for patients' outcome than TS IHC.     

Development of a nested PCR assay for the detection of Fusarium solani DNA and its evaluation in the diagnosis of invasive fusariosis using an experimental mouse model

Fusarium infections are increasingly being encountered in immunocompromised patients. Fusarium solani accounts for nearly half of these infections. A specific nested PCR (nPCR) assay has been developed by using DNA isolated from several Fusarium species and other common fungi. Furthermore, DNA samples isolated from bronchoalveolar lavage (BAL) and serum samples from mice infected intravenously with F. solani conidia and sacrificed on every third day post infection were used for the evaluation of the established nPCR protocol. The lung homogenate, BAL and blood from infected animals were also cultured. The nPCR assay was specific for F. solani and detected 450 fg of DNA corresponding roughly to 11 F. solani cells. Cultures of lung homogenate of infected animals up to day 16 yielded F. solani with decreasing fungal load and were negative thereafter. The nPCR positivity in BAL was 100% concordant with lung tissue culture results. Although detection of F. solani DNA in serum was less sensitive than in BAL, it remained positive for longer duration. Our data from an experimental mouse model show that detection of DNA in BAL and to a lesser extent in serum by nPCR offers a sensitive and specific diagnostic approach to invasive F. solani infection.     

Comprehensive mutational scanning of the p53 coding region by two- dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long- distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li- Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.      

DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach

Clonal T-cell receptor gamma (TCR-gamma) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-gamma rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vgamma segments in two distinct reactions either with a Jgamma1.3/2.3 or a Jgamma1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VgammaI-Jgamma1.3/2.3 including 51 biclonal rearrangements. For characterisation of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jgamma primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-gamma rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.     

食管癌与胸苷酸合成酶多态性的关联研究

目的探讨胸苷酸合成酶（TS）5’非编码区（UTR）串联重复序列和3’UTR6bp缺失或插入多态性对食管鳞状细胞癌（ESCC）的发生及淋巴结转移的作用。方法从232例ESCC患者和348例健康对照者的外周血中提取白细胞DNA,分别用PCR-片段长度和PCR—RFLP方法检测TS 5’UTR和3’UTR基因型。结果TS5’UTR、3’UTR的基因型和等位基因型分布在ESCC患者与健康对照者中,差异无统计学意义,它们不单独作用于ESCC的发生和发展。但这两种多态性联合分析显示,同时携带3R／3R及6bp＋／6bp＋基因型的个体,其患ESCC的危险性显著低于携带其他基因型组合的个体（校正OR=0．32,95％CI=0．08～0．92）。5’UTR 2R／3R基因型显著增加ESCC淋巴结转移的危险性（校正OR=3．68,95％CI=1．54—8．93）。结论TS5’UTR重复序列多态性和3’UTR缺失多态性联合分析可作为预测ESCC易感性的标志,而5’UTR 2R／3R可作为预测ESCC淋巴结转移的候选分子指标。     

Comparison of a quantitative PCR method with FISH for the assessment of the four aneuploidies commonly evaluated in CLL patients.

Four chromosomal defects associated with outcome are commonly evaluated by fluorescent in situ hybridization (FISH) in chronic lymphocytic leukemia (CLL), namely deletions of the 13q13-q14, 11q22 and 17p13 regions and trisomy 12. In this study, we compared a quantitative PCR method--quantitative multiplex PCR of short fluorescent fragment (QMPSF)--with FISH for the detection of these acquired aneuploidies in a series of 110 patients with Binet stage A CLL. Genes located in the deleted or gained regions were selected as target genes and amplified using a method based on the simultaneous amplification of short fluorescent genomic fragments under quantitative conditions. A chromosomal imbalance involving one or several of the four loci was detected by either method in 72 patients (65%). A chromosome 13 deletion was present in 61 patients (54%), a 11q22 deletion in nine (8%), a trisomy 12 in nine and a 17p deletion in one. FISH and QMPSF results were identical for 103 out of 110 patients and discrepancies could be explained in most cases. This study demonstrates that a quantitative multiplex PCR represents a cost-effective method that could replace FISH in CLL patients. However, although QMPSF is perfectly adapted to the detection of primary defects, care should be taken when searching for clonal evolutions present in a small proportion of tumor cells.     

Evaluation of multiplex real‐time PCR for identifying dermatophytes in clinical samples—A multicentre study

The gold‐standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real‐time polymerase chain reaction (RT‐PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT‐PCR was further evaluated in 200 community‐based samples obtained from a health maintenance organisation and 103 military‐personnel‐based samples analysed at a central laboratory. In hospital‐based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non‐concordance, sequencing was consistent with RT‐PCR results. RT‐PCR correctly identified all smear‐ and culture‐positive cases in community and military‐personnel samples. The results were available within 4 hours. The multiplex RT‐PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.     

银染RNA AP-PCR差示法克隆人胃癌转移相关基因

目的:以改进的银染RNAAP-PCR法分析原发性及转移性胃癌标本,鉴定和克隆胃癌转移相关基因。方法:以原发及转移灶胃癌标本总RNA为研究对象,首先以T12MN“锚定”引物进行逆转录合成cDNA第一链,再以10核苷酸任意引物进行PCR扩增,5%非变性聚丙烯酰胺凝胶电泳分离扩增产物后银染显示并回收差异条带,经RNA点杂交验证、克隆测序后进入GenBank数据库检验同源性。结果:经银染RNAAP-PCR法分析,获得系列差异表达片段。对其中MGA1(662bp)和PGA1(589bp)两个片段进行验证和测序,结果显示,MGA1与胃癌转移相关,并与编码人巨噬细胞集落刺激因子受体(macrophagecolony-stimulatingfactorreceptor,CSF-1R)的原癌基因c-fms100%同源;而PGA1则属胃癌转移抑制基因,与编码肿瘤转移抑制基因KAI-1完全同源。结论:银染RNAAP-PCR法适用于分析和克隆差异表达基因,具有快速、直观、假阳性率低等优点;提示CSF-1R及KAI-1与胃癌转移表型相关,为进一步研究CSF-1R功能提供了新的线索。     

急性早幼粒细胞白血病PML/RARα融合基因筛查及实时定量检测平台的建立

目的：针对90％以上的急性早幼粒细胞白血病（acute promyelocytic leukemia,APL）患者表达的PML／RARc~融合基因,设计引物及探针。对APL患者进行基因筛查,并构建PMI／RARα环形质粒作为标准品,建立APL患者PML／RARα融合基因检测及微小残留病变监测的诊断平台,为APL患者的诊治提供分子生物学依据。方法：设计PML／RARα及ABL引物及Taqman探针,对APL患者进行基因筛查。并以PML／RARα L型及S型阳性的APL患者cDNA为模板,应用PCR技术扩增出453bp和550bp基因片段,构建pMD18T—PML／RARα（L）及pMD18T—PML／RARα（s）标准品。实时荧光定量（real—time quantitative PCR,RQ—PCR）技术对该基因转录本水平的变化情况进行监测。结果：成功构建pMD18T—PML／RARα质粒标准品,应用RQ—PCR技术,以ABL为内参,应用Taqman探针法,对APL患者标本进行检测,技术可行,数据稳定。结论：成功构建APL融合基因检测及微小残留病变监测的诊断平台,应用于临床病人的基因诊断,为APL的诊治提供了可靠的分子依据。     

Serotype identification of Cryptococcus neoformans by multiplex PCR

The pathogenic yeast Cryptococcus neoformans is traditionally classified into three varieties with five serotypes: var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C) and serotype AD (hybrid of serotypes A and D). A commercial kit, Crypto Check (Iatron Laboratories, Tokyo, Japan), has been used worldwide for serotyping isolated strains. However, its production was discontinued in 2004, and hence the present study aimed to develop a simple polymerase chain reaction (PCR) method for serotyping C. neoformans strains. Subjecting genomic DNA of 59 strains of the five serotypes to multiplex PCR amplification using a set of four primers designed for the laccase gene (LAC1) differentiated serotypes A, D, B and C, but could not separate serotype AD from serotype D. However, a primer pair designed for the capsule gene (CAP64) allowed serotypes D and AD to be differentiated. When PCR amplification was performed in the simultaneous presence of the above six primers, the five serotypes produced two to five DNA fragments that could be used to distinguish them. This multiplex PCR method is useful for serotyping C. neoformans isolates, and represents an effective replacement for the Crypto Check kit.     

A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings.

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and beta-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach.     

多重巢式RT-PCR和染色体核型分析技术在急性髓系白血病中的联合应用

背景与目的:细胞遗传学分析在白血病的诊断和预后判断中有重要价值,但常规显带不仅耗时,且难以获得良好的分裂相;而聚合酶链反应具有灵敏、快速的特点.本研究探讨联合应用多重巢式RT-PCR和染色体核型分析,对急性髓系白血病(acute myeloid leukemia,AML)中融合基因的表达及其在各亚型的分布和克隆性染色体异常的检出情况.方法:采用多重巢式RT-PCR技术对60例AML病例进行检测,其中37例同时进行染色体R或G显带.结果:60例AML患者中检出融合基因28例(46.7%),包括AML1/ETO、PML/RARα、CBFβ/MYH11、MLL基因异常(包括MLL/AF6、MLL/AF9、MLL/AF10、MLL/MLL)、DEK/CAN、TEL/PDGFR、AML1/MDS1(EVI-1).同时进行染色体R或G显带的37例病例中有30例可供分析,其中14例(46.7%)检出染色体结构和数目异常;联合多重RT-PCR可使AML克隆性染色体异常检出率增至59.5%(22/37).结论:联合多重巢式RT-PCR和染色体核型分析技术可以提高克隆性染色体异常的检出率.     

Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction.

The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp.     

Multiplex PCR for the detection of BCL-1/IGH and BCL-2/IGH gene rearrangements--clinical validation in a prospective study of blood and bone marrow in 258 patients with or suspected of non-Hodgkin's lymphoma

We have designed a multiplex PCR, which allows for fast and high throughput demonstration of the BCL-1/IGH and BCL-2/IGH fusion DNA observed primarily in mantle cell- and follicular non-Hodgkin's lymphoma (NHL). Blood (PB) and/or bone marrow (BM) from 258 patients suspected of NHL have prospectively been evaluated. Eleven patients (4%) were found t(11;14)+ and 37 patients (14%) t(14;18)+. Comparing these results to standard diagnostic methods of PB and/or BM identified PCR+ samples that were normal by morphology (BCL-1/IGH: 1/11; BCL-2/IGH: 17/37). Equally important, patients who were not clonal in PB and/or BM by flow cytometry were identified as PCR+ (BCL-1/IGH: 3/11; BCL-2/IGH: 23/37). We conclude that this multiplex approach allows for easy and sensitive molecular determination of molecular lesions in NHL, which have diagnostic and prognostic importance.