Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinert's disease)

Myotonic dystrophy (DM), or Steinert's disease, is an autosomal dominant disease characterized by myotonia, muscular weakness and atrophy, as well as lens opacities, cardiomyopathy and mild endocrine changes. The gene for DM located on 19q contains a triplet repeat at the 3' end of the gene. In DM patients, this repeat is found to be expanded. We have previously described a preimplantation genetic diagnosis (PGD) for DM using polymerase chain reaction (PCR) followed by conventional analysis on ethidium bromide-stained gels. The major drawback of this system was that allelic dropout occurred in >20% of the cells, leading to the loss of healthy embryos for transfer. To resolve this problem, we developed a PGD for DM using fluorescent PCR followed by fragment analysis on an automated DNA sequencer and made a comparison between the conventional PCR described earlier and fluorescent PCR, which turned out to be superior in accuracy and efficiency. Three PGD cycles were performed using fluorescent PCR and are described here.      

Real-time polymerase chain reaction and its potential clinical application

The review introduces real-time polymerase technique, a modern high-tech method, to the medical community. Physico-chemical principles of the method, its main stages, and variants of techniques applying fluorescent reporter platforms are considered. Equations used for quantitative estimation, methods of data processing are given; absolute and relative quantification of specific targets is considered. The special part of the review is dedicated to clinical application of the method by the example of infectious and oncological diseases.     

Effectiveness of rapid multiplex polymerase chain reaction for early diagnosis and treatment of pertussis

BackgroundPertussis, is an infectious respiratory disease caused by Bordetella pertussis. The incidence of pertussis has been increasing in South Korea to due to waning vaccine-induced immunity. Culture has a low sensitivity and a long turnaround time (TAT). Recently, a rapid multi-polymerase chain reaction (mPCR) test with a TAT of about 1 h was developed for the detection of respiratory pathogens (17 viruses and three bacteria), including B. pertussis. This study aimed to investigate the effectiveness of mPCR for early diagnosis and treatment of pertussis.MethodsWe performed a retrospective study of patients with pertussis diagnosed from May 2017 to June 2019 at a university hospital in South Korea. Nasopharyngeal swab specimens were tested using mPCR. Data were extracted from medical records.ResultsA total of 27 patients with a median age of 48.9 years (range: 3.3–82.2 years) were diagnosed with pertussis, of whom 9 (33.3%) were male. Eleven (40.7%) had fever, 12 (44.4%) had dyspnea, three (11.1%) had paroxysmal cough, and nine (33.3%) had inspiratory whooping. The median interval from symptom onset to diagnosis was 9.0 days (range: 1–31 days). Twenty-four patients (81.5%) were diagnosed within 2 weeks from symptom onset. All but one patient was prescribed macrolide antibiotics. Twenty-two patients (81.5%) required hospitalization, including three (11.1%) who required intensive care unit care for ventilation.ConclusionTesting patients with respiratory symptoms using mPCR can improve early diagnosis of pertussis, ensure proper treatment, and may help with outbreak control.     

应用巢式PCR对单细胞进行性别诊断的初步研究

目的：应用巢式PCR技术对人类植入前胚胎进行性别诊断。方法：收集单个或两个淋巴细胞和卵裂球（50个／组），按不同的方法处理单细胞（纯水法、冻融法、碱法），而后行巢式PCR扩增牙釉质基因。结果：纯水法、冻融法、碱法处理后，扩增率分别为83％、94％、95％。后两种处理方法的扩增效率明显高于纯水法（P＜0．01），通过检测正常男性单淋巴细胞基因型发现3种方法等位基因脱失率分别为24％、12％、4％，差异有显著性（P＜0．05）。两个淋巴细胞或卵裂球的扩增率及等位基因脱失率与单细胞相比差异无显著性。结论：提高植入前遗传学诊断的准确性主要取决于如何克服单细胞PCR的缺点，采用碱法裂解单细胞及取两个卵裂球进行检测可提高用于性别诊断的单细胞PCR技术准确性和敏感性。     

FISH应用于植入前遗传学诊断对高风险胚胎检测的研究

目的对固定好的单卵裂球进行FISH检测,在最短的时间内获得清晰可靠的信号,进行临床分析.方法选取发育不良的高风险胚胎分别进行分开变性和共变性后杂交的单细胞FISH,统计并分析其信号结果.结果共变性法信号获得率较分开变性法为高,并且信号获得率与变性方法相关(P=0.0486);共有9枚胚胎,42个卵裂球固定良好,其中二倍体信号36个,单倍体信号2个,21号染色体异常的胚胎为4︰9.结论初探分析人IVF废弃胚胎21号染色体异常几率较高.     

HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis

CONTEXT: Hereditary hemochromatosis is recognized as one of the most common autosomal recessive disorders, with a prevalence of 1 in 200 to 400 in the white population. Early detection and treatment are completely effective in preventing pathology. It is anticipated that testing for hereditary hemochromatosis will increase, as will the need for a technology that can handle the demand. OBJECTIVE: To describe a high-throughput, single-tube, allele-specific multiplex polymerase chain reaction assay for identifying the 2 mutations in the HFE gene associated with hereditary hemochromatosis. DESIGN: Fluorescence-labeled polymerase chain reaction products from a multiplex polymerase chain reaction are analyzed by automated capillary electrophoresis. DATA ANALYSIS: The assay was validated by analysis of 25 blinded samples, and results were concordant with an established laboratory assay. CONCLUSION: The assay described offers a significant improvement over manual laboratory assays in throughput, reduced technologist time, and cost.     

Development and application of a novel multiplex polymerase chain reaction for semi-quantitation of human Cytomegalovirus in clinical specimens

Human Cytomegalovirus (HCMV) is an important cause of morbidity and occasional mortality in immunocompromised patients. The aims of the study were to develop and apply a multiplex PCR for semi-quantitation of HCMV in clinical specimens, compare its efficiency with pp65 antigenemia assay and real-time PCR. A multiplex PCR combining the primers targeting three regions of the HCMV genome, viz. the morphological transforming region II (mtr II), UL-83 and glycoprotein O (gO) genes for the detection of the genome of HCMV was standardized with HCMV AD169 strain. This was evaluated against pp65 antigenemia assay by applying it on 70 peripheral blood specimens obtained from 70 post-renal transplant recipients. The multiplex PCR and a real-time PCR were prospectively applied to 31 clinical specimens from 29 immunocompromised patients. The multiplex PCR was specific for HCMV. The level of antigenemia and the copy number of the viral DNA as estimated by real-time PCR in the samples positive for all the three targets was significantly higher than in those that were positive for only one or two of the targets. The multiplex PCR provides a simple and effective means of quantifying HCMV in clinical specimens with efficiency equivalent to the pp65 antigenemia assay and real-time PCR.     

The utility of multiplex polymerase chain reaction for diagnosis of infectious diarrhoea in a tropical country

PurposeThe etiology of infective diarrheaoften remains undiagnosed. We studied the role of multiplex polymerase chain reaction (PCR) for detection of etiological agents of diarrhoea.MethodsFast track diagnostics (FTD)gastroenteritis panel for bacterial and viral pathogens was used to test stool samples from patients with diarrhoea.ResultsStool samples from 276 patients (138 immunocompetent and 138 immunocompromised) with diarrhoea and 138 healthy controls were tested. Bacterial culture was positive in 5 samples. Following agents were isolated: Shigella sonnei(2), Shigella dysentriae(1), SalmonellaParatyphi B(1) and Vibrio cholerae (1). Multiplex PCR panel did not include Vibrio cholerae in its panel. A total of 65 target pathogens were identified in 60/276 (21.7%) patients by multiplex PCR. 28/65(41.1%) and 37/65 (56.9%) were bacterial and viral agents respectively. Identified bacteria were Shigella(20), Salmonella(3), Campylobacter(4) and Clostridium difficile(1). Viral targets identified were Norovirus GII (28), Adenovirus(4), Astrovirus(3) and Sapovirus(2). All the controls were negative for enteropathogens by conventional methods and multiplex PCR.ConclusionsOur detection rates increased from 1.8% (5/276)by conventional methods to 21.7% (60/276)by multiplex PCR, which included both bacterial as well as viral targets.     

Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells

We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1–110 mutation leading to beta-thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF-PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri-allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF-PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.     

Clinical application of multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies

The clinical application of quantitative fluorescent polymerase chain reaction (QF-PCR) for rapid prenatal detection of chromosome aneuploidies has been limited in most studies to the detection of autosomal trisomies. Recently it has been shown that a newly identified highly polymorphic marker, termed X22, which maps to the Xq/Yq pseudoautosomal region of the sex chromosomes, used together with the X-linked short tandem repeat (STR) HPRT, allows the accurate detection of gonosome aneuploidies. We have developed a rapid assay, which includes these STR markers together with a sequence of the amelogenin region of the sex chromosomes and selected highly polymorphic autosomal STR. Two more X chromosome markers, as yet not used in previous QF-PCR applications, were also included in the assay. The molecular test was then used in a clinical trial on 551 uncultured amniotic fluid samples, allowing the assessment of copy number for chromosomes X, Y and 21 in 100% of cases. In the course of this study, two fetuses with Turner's syndrome and one with Klinefelter's syndrome were identified along with 17 autosomal trisomies. The assay proved to be so efficient and reliable that in most aneuploidy cases, in which ultrasound findings were in agreement with the molecular result, therapeutical interventions were possible without waiting for the result of cytogenetic analysis.     

Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos

Cultured human preimplantation embryos have been used to developmethods which allow preimplantation genetic diagnosis (PGD)analyses by polymerase chain reaction (PCR) and fluorescentin-situ hybridization (FISH) on biopsied blastomeres and trophectodermcells from the same embryo. An experimental design is describedand experiments undertaken, which demonstrate the feasibilityof extending biopsy and PGD procedures currently in use. Wehave shown that dual-stage biopsies are possible, and that thePCR and FISH analyses of the biopsied cell samples are effective.One to two blastomeres were biopsied from an 8- to 10-cell embryoand processed for the simultaneous PCR amplification of a -globinand a cytosine adenine (CA) repeat sequence, or a Y chromosomesequence. FISH procedures were also used to detect the presenceof Y chromosome markers. The biopsied cleavage-stage embryocan be cultured to the blastocyst stage, where the serial biopsyof three to five mural trophectoderm cells provides two furthercell samples. These can be used to repeat and/or undertake additionalPGD analyses. The biopsied blastocyst is either used to confirmearlier diagnoses, or placed in culture for a further 4–24h. Maintenance of a blastocoele cavity, hatching and formationof an outgrowth demonstrates continuing viability followingthe dual-stage biopsy procedures. The PCR DNA amplificationprocedures are effective at the cellular level for both biopsiedblastomeres and mural trophectoderm cells. The FISH techniqueshave shown a definitive Y signal in 50% (one out of two) and100% (two out of two) of the biopsied blastomeres and 72% (twoout of three, four out of five and 7 out of 10) for the trophectodermcell nuclei. Preliminary experiments have demonstrated thatthe FISH preparations can be re-amplified to improve the signal,and dual fluorescent procedures using the X and Y probes areeffective. A retrospective PCR analysis has also been undertakenon preparations of biopsied cells which were previously usedfor PGD analysis by FISH.     

内含子或外显子缺失DMD家系女性携带者的基因诊断

目的对内含子和(或)外显子缺失的Duchenne型肌营养不良症(Duchenne muscular dystrophy,DMD)家系女性成员进行致病基因携带者的诊断,为进一步行产前诊断或植入前遗传学诊断提供准确的信息。方法利用dystrophin基因5个微卫星位点(STR-44、45、49、40、5′DysⅡ)的多态性连锁分析,同时结合半定量PCR对1个内含子和外显子均缺失的DMD家系(家系5)和1个仅内含子缺失的DMD家系(家系4)的女性成员进行携带者基因诊断。结果家系5的Ⅱ2的STR-50位点基因型为245/245,不是DMD基因携带者。家系4的Ⅱ6、Ⅱ8的STR-45位点基因型为del/172,Ⅲ19为del/178,均为DMD基因携带者。结论STR-PCR多态性分析结合内含子半定量PCR可获得更多诊断信息,更准确地检出内含子缺失的女性携带者,提高诊断率。     

孕妇外周血中胎儿短串联重复序列基因型的检测

目的探讨利用孕妇血浆中胎儿DNA进行遗传病产前基因诊断的可行性。方法应用9个具有高度多态性的短串联重复序列（short tandem repeat，STR）位点，以多重荧光PCR方法对10名健康孕妇孕早、中、晚期共30份血浆标本和非孕期血浆标本中DNA进行STR等位基因扩增。同时检测孕妇丈夫的外周血本，然后进行基因扫描及对照分析，判断胎儿父源性等位基因在孕妇血浆中的检出情况。结果10名孕妇均足月分娩，男婴3名，女婴7名。10名孕妇血浆中均检出胎儿父源性等位基因，即胎儿DNA。30份孕期血浆标本中有23份检出胎儿DNA，其中孕早期标本6份（6／10）；孕中期标本8份（8／10）；孕晚期标本9份（9／10）。7份标本未见到胎儿DNA。结论应用多重荧光PCR方法对孕妇血浆中DNA进行SFR多态化点的复合扩增，可获得男性和女性胎儿DNA信息，可用于无创伤性产前诊断。     

全染色体涂抹探针在女性罗伯逊易位携带者植入前遗传学诊断中的临床应用

目的应用全染色体涂抹探针（whole chromosome painting probe，WCP）对女性罗伯逊易位携带者进行卵母细胞第一极体的植入前遗传学诊断（preimplantation genetic diagnosis，PGD）。方法应用全染色体涂抹探针进行第一极体荧光原位杂交，对4例女方罗伯逊易位携带者进行了4个周期的PGD。患者染色体核型均为45，XX，der（13；14），（q10；q10）。所有周期取卵后6h内通过活检取出第一极体，采用WCP探针进行荧光原位杂交，受精后第3天选择染色体组成正常或平衡的胚胎进行宫腔内移植。结果4个周期共获卵61个，其中54个成熟可进行活检，活检成功率92．6％（50／54），固定成功率90．O％（45／50）。40个获得明确诊断，总体诊断率为74．1％（40／54）。卵胞浆内单精子注射后受精率64．8％（35／54），优质胚胎率为65．7％（23／35）。获得2例临床妊娠。其中1例于孕9周胚胎停止发育，绒毛染色体分析核型为45，X；另1例产前诊断证实核型为46，XX。2006年6月足月分娩一正常活女婴。结论全染色体涂抹探针可准确区分正常、平衡以及异常卵子，从而可有效应用于女性染色体易位携带者的PGD。     

Design and validation of a multiplex specific primer-directed polymerase chain reaction assay for killer-cell immunoglobulin-like receptor genetic profiling

Current methodologies for the analysis of the killer-cell immunoglobulin-like receptor (KIR) locus utilize specific primer-directed polymerase chain reaction (SSP-PCR), which require a wide range of DNA input, multiple reaction conditions, and up to 16 individual reactions. We have developed and validated a multiplex SSP-PCR method for the genetic analysis of the KIR locus. Design and optimization of four multiplex groups targeting 14 genes and their alleles on the KIR locus has been completed. Each multiplex group contains PCR products that differ in size by a minimum of 15 bp to allow sufficient fragment length resolution for size discrimination by gel electrophoresis. This assay allows for efficient genotyping of the KIR locus while requiring a minimum amount of DNA input, utilizing the simplicity of SSP-PCR.