Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.     

Plasma choline-containing phospholipids: potential biomarkers for colorectal cancer progression

Colorectal cancer (CRC) is believed to progress through the adenoma–carcinoma sequence. The adenoma–carcinoma transition is an important window for early detection and intervention of CRC. In the present study, plasma samples from patients with CRC (n = 120), patients with adenomatous polyps (AP) (n = 120), and healthy controls (n = 120) were collected. Plasma phospholipid levels were analyzed with liquid chromatography–tandem mass spectrometry. It was found that the plasma levels of major lysophosphatidylcholine (LPC) species were gradationally decreased from healthy controls, AP to CRC subjects. A formula including total saturated LPCs, 18:2 LPC and sphingosylphosphorylcholine (SPC) yielded a sensitivity and specificity of 88.3 and 80 % for separating CRC from healthy controls. An optimized model with total saturated LPCs, 20:4 LPC and sphingomyelins (SM) as markers yielded a sensitivity and specificity of 89 and 80 % for separating AP from the healthy controls. Moreover, with SM, SPC and saturated LPCs as markers, a model was made to separate CRC from AP with the sensitivity and specificity of 90 and 92.5 %, respectively. These data indicate that the plasma choline-containing phospholipid levels represent potential biomarkers to distinguish between healthy controls, AP and CRC cases, implying their clinical usage in CRC and/or AP-CRC progression detection.     

A panel of three plasma microRNAs for colorectal cancer diagnosis

BackgroundDifferential microRNA (miRNA) expression profiles in plasma or serum were identified, providing foundation for studying their potentially diagnostic role in colorectal cancer (CRC).MethodsWe performed S-poly(T) Plus PCR assay to select and validate differentially expressed plasma miRNAs from a sample set including 101 CRC patients, 20 patients with colorectal noncancerous polyps (NCP), and 134 healthy controls. And bioinformatics methods was used to integrated predicted or validated targets of the differentially dysregulated miRNAs and analyzed their overrepresented pathways.ResultsAfter the two-phase selection and validation process, we identified a miRNA panel (miR-144-3p, miR-425-5p, and miR-1260b) with high diagnostic efficiency for CRC; the panel distinguished CRC patients from controls with 93.8% sensitivity and 91.3% specificity. Results indicated that the dysregulated miRNAs in CRC were functionally involved in several key cancer-related pathways, such as axonal guidance, PI3K, and calcium signaling pathways.ConclusionsOur study demonstrated that a plasma 3-miRNA panel may serve as a novel noninvasive biomarker to diagnose CRC. This plasma 3-miRNA panel may be related to CRC development. However, further studies are needed to highlight its theoretical strengths.     

Identification of Gene-Expression Signatures and Protein Markers for Breast Cancer Grading and Staging

The grade of a cancer is a measure of the cancer''s malignancy level, and the stage of a cancer refers to the size and the extent that the cancer has spread. Here we present a computational method for prediction of gene signatures and blood/urine protein markers for breast cancer grades and stages based on RNA-seq data, which are retrieved from the TCGA breast cancer dataset and cover 111 pairs of disease and matching adjacent noncancerous tissues with pathologists-assigned stages and grades. By applying a differential expression and an SVM-based classification approach, we found that 324 and 227 genes in cancer have their expression levels consistently up-regulated vs. their matching controls in a grade- and stage-dependent manner, respectively. By using these genes, we predicted a 9-gene panel as a gene signature for distinguishing poorly differentiated from moderately and well differentiated breast cancers, and a 19-gene panel as a gene signature for discriminating between the moderately and well differentiated breast cancers. Similarly, a 30-gene panel and a 21-gene panel are predicted as gene signatures for distinguishing advanced stage (stages III-IV) from early stage (stages I-II) cancer samples and for distinguishing stage II from stage I samples, respectively. We expect these gene panels can be used as gene-expression signatures for cancer grade and stage classification. In addition, of the 324 grade-dependent genes, 188 and 66 encode proteins that are predicted to be blood-secretory and urine-excretory, respectively; and of the 227 stage-dependent genes, 123 and 51 encode proteins predicted to be blood-secretory and urine-excretory, respectively. We anticipate that some combinations of these blood and urine proteins could serve as markers for monitoring breast cancer at specific grades and stages through blood and urine tests.     

Simple Detection of the IS6110 Sequence of Mycobacterium tuberculosis Complex in Sputum,Based on PCR with Graphene Oxide

Graphene oxide (GO) has proven to be a satisfactory DNA-sensor platform for applications in enzyme-free signal amplification, fluorescence-based amplification, and nanoparticle-based platforms because of its excellent electrical, thermal, and optical properties. In this study, we designed a novel platform for the fluorescence detection of biomolecules, using a fluorescent dye-labeled primer and GO. We applied this system for the detection of the IS6110 insertion sequence of the Mycobacterium tuberculosis complex (MTB) and evaluated its feasibility for use in molecular diagnostics. Fifty-four sputum specimens were collected at our institution from October 2010 to March 2012. To detect MTB in the samples, we performed PCR amplification of the IS6110 DNA sequence using FAM-labeled primers, after which the PCR amplicon was incubated with GO and the fluorescence was measured. The results were compared with those obtained by conventional real-time quantitative PCR (RQ-PCR). The fluorescence intensity observed increased in a concentration-dependent manner with the FAM-labeled IS6110 amplicon. The results of the PCR-GO system for detecting IS6110 DNA were in good agreement with those obtained with conventional RQ-PCR (kappa statistic = 0.925). The PCR-GO system detected MTB DNA in 23 of 25 RQ-PCR-positive sputum samples (92.0%; 95% CI, 75.0–98.0%), but not in 29 of 29 RQ-PCR-negative sputum samples (100%; 95% CI, 88.1–100.0%). These results indicate the utility of the PCR-GO system in molecular diagnostics.     

A gene marker panel covering the Wnt and the Ras-Raf-MEK-MAPK signalling pathways allows to detect gene mutations in 80% of early (UICC I) colon cancer stages in humans

Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.     

Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2, SDC2, and PRIMA1 in plasma samples

Aberrant methylation is one of the most frequent epigenetic alterations that can contribute to tumor formation. Cell-free DNA can originate from tumor tissue; therefore, the evaluation of methylation markers in cell-free DNA can be a promising method for cancer screening. Our aim was to develop a panel of biomarkers with altered methylation along the colorectal adenoma-carcinoma sequence in both colonic tissue and plasma. Methylation of selected CpG sites in healthy colonic (n = 15), adenoma (n = 15), and colorectal cancer (n = 15) tissues was analyzed by pyrosequencing. MethyLight PCR was applied to study the DNA methylation of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters in 121 plasma and 32 biopsy samples. The effect of altered promoter methylation on protein expression was examined by immunohistochemistry. Significantly higher (P < 0.05) DNA methylation levels were detected in the promoter regions of all 4 markers, both in CRC and adenoma tissues compared with healthy controls. Methylation of SFRP1, SFRP2, SDC2, and PRIMA1 promoter sequences was observed in 85.1%, 72.3%, 89.4%, and 80.9% of plasma samples from patients with CRC and 89.2%, 83.8%, 81.1% and 70.3% from adenoma patients, respectively. When applied as a panel, CRC patients could be distinguished from controls with 91.5% sensitivity and 97.3% specificity [area under the curve (AUC) = 0.978], while adenoma samples could be differentiated with 89.2% sensitivity and 86.5% specificity (AUC = 0.937). Immunohistochemical analysis indicated decreasing protein levels of all 4 markers along the colorectal adenoma-carcinoma sequence. Our findings suggest that this methylation biomarker panel allows non-invasive detection of colorectal adenoma and cancer from plasma samples.     

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum

Aims:  To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Methods and Results:  Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin ( bont ) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A , bont/B , bont/E , bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 10³ to 10⁴ cells ml⁻¹. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.
Conclusion:  These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Significance and Impact of the Study:  Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 10(4) cells at 29 cycles and over 10(5) cells at 25 cycles. In contrast, the SPD-based CE-LIF has revealed 6-base repeats in samples with as low as 10(2) cells after 29 cycles and 10(3) cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.     

Evaluation of the Cepheid GeneXpert system for detecting Bacillus anthracis

AIMS: The Cepheid GeneXpert is a four-site, automated sample preparation and real-time PCR detection system. In this study, the capability of the GeneXpert to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. METHODS AND RESULTS: A four-plex, dried-down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross-reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross-reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert to those run directly on the Cepheid SmartCycler without sample processing. The GeneXpert detection capability was three logs lower than the SmartCycler indicating the benefit of incorporating a nucleic acid extraction procedure. CONCLUSIONS: This study demonstrates that the GeneXpert is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. SIGNIFICANCE AND IMPACT OF THE STUDY: The GeneXpert is the only platform currently available that is capable of both nucleic acid purification and real-time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.     

Detection of Mycoplasma pneumoniae in simulated and true clinical throat swab specimens by nanorod array-surface-enhanced Raman spectroscopy

The prokaryote Mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The limitations of existing options for mycoplasma diagnosis highlight a critical need for a new detection platform with high sensitivity, specificity, and expediency. Here we evaluated silver nanorod arrays (NA) as a biosensing platform for detection and differentiation of M. pneumoniae in culture and in spiked and true clinical throat swab samples by surface-enhanced Raman spectroscopy (SERS). Three M. pneumoniae strains were reproducibly differentiated by NA-SERS with 95%-100% specificity and 94-100% sensitivity, and with a lower detection limit exceeding standard PCR. Analysis of throat swab samples spiked with M. pneumoniae yielded detection in a complex, clinically relevant background with >90% accuracy and high sensitivity. In addition, NA-SERS correctly classified with >97% accuracy, ten true clinical throat swab samples previously established by real-time PCR and culture to be positive or negative for M. pneumoniae. Our findings suggest that the unique biochemical specificity of Raman spectroscopy, combined with reproducible spectral enhancement by silver NA, holds great promise as a superior platform for rapid and sensitive detection and identification of M. pneumoniae, with potential for point-of-care application.     

应用TaqMan荧光定量PCR检测土拉弗朗西斯菌

目的：利用Roche LightCycler实时定量PCR系统建立一种快速、灵敏、特异的检测土拉弗朗西斯菌的方法。方法：基于TaqMan荧光探针实时定量PCR技术,选择土拉弗朗西斯菌染色体上的特异序列[醇醛酮还原酶（AKR）和外膜蛋白FopA基因]作为检测靶序列,建立土拉弗朗西斯菌实时定量PCR检测方法;评价该检测方法的特异性和灵敏性;采用克隆菌株污染环境土壤来模拟实际样品,评价该检测方法在快速检测与现场检测等实际应用中的表现。结果：优化筛选基因组中的FT-AKR和FT-fopA片段作为检测靶序列,所建立的土拉弗朗西斯菌实时定量PCR检测方法检测克隆菌株质粒的灵敏度均为10个拷贝/每个反应体系;以其他非土拉弗朗西斯菌为模板未出现非特异扩增;模拟环境土壤样品检测灵敏度2个引物对分别为440和960CFU/g土壤;盲测实验结果显示对于灵敏度范围内的阳性样本均能正确识别,并能正确检测出不同浓度的阳性样本。以FT-fopA片段为靶序列的扩增效率不及基于FT-AKR引物对的扩增。结论：基于FT-AKR片段的引物对扩增效率高,检测土拉弗朗西斯菌具有特异、灵敏的特点,对临床诊断、环境污染监测、防治生物突发事件等具有重要意义。     

Simultaneous detection of waterborne viruses by multiplex real-time PCR

Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex real-time PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.     

Blood-Based Biomarkers of Aggressive Prostate Cancer

Purpose

Prostate cancer is a bimodal disease with aggressive and indolent forms. Current prostate-specific-antigen testing and digital rectal examination screening provide ambiguous results leading to both under-and over-treatment. Accurate, consistent diagnosis is crucial to risk-stratify patients and facilitate clinical decision making as to treatment versus active surveillance. Diagnosis is currently achieved by needle biopsy, a painful procedure. Thus, there is a clinical need for a minimally-invasive test to determine prostate cancer aggressiveness. A blood sample to predict Gleason score, which is known to reflect aggressiveness of the cancer, could serve as such a test.

Materials and Methods

Blood mRNA was isolated from North American and Malaysian prostate cancer patients/controls. Microarray analysis was conducted utilizing the Affymetrix U133 plus 2·0 platform. Expression profiles from 255 patients/controls generated 85 candidate biomarkers. Following quantitative real-time PCR (qRT-PCR) analysis, ten disease-associated biomarkers remained for paired statistical analysis and normalization.

Results

Microarray analysis was conducted to identify 85 genes differentially expressed between aggressive prostate cancer (Gleason score ≥8) and controls. Expression of these genes was qRT-PCR verified. Statistical analysis yielded a final seven-gene panel evaluated as six gene-ratio duplexes. This molecular signature predicted as aggressive (ie, Gleason score ≥8) 55% of G6 samples, 49% of G7(3+4), 79% of G7(4+3) and 83% of G8-10, while rejecting 98% of controls.

Conclusion

In this study, we have developed a novel, blood-based biomarker panel which can be used as the basis of a simple blood test to identify men with aggressive prostate cancer and thereby reduce the overdiagnosis and overtreatment that currently results from diagnosis using PSA alone. We discuss possible clinical uses of the panel to identify men more likely to benefit from biopsy and immediate therapy versus those more suited to an “active surveillance” strategy.