Detection of the phosphorothioate oligonucleotide fomivirsen using a ligase detection reaction with polymerase chain reaction.

This study aimed to develop a simple and sensitive detection method for fomivirsen, a 21-nucleotide phosphorothioate oligonucleotide used as a nucleic acid medicine, using a ligase detection reaction. A ligation probe was designed to hybridize with fomivirsen and polymerase chain reaction (PCR) primers, with a deoxyuridine part between the primer binding sites. The probe was ligated to a circular product by Taq DNA ligase, and the resulting product was converted to a linear form through the removal of the uracil base using uracil DNA glycosylase. The linear product was then quantified using real-time PCR. The developed method could detect 0.025-6.4 nM of fomivirsen in water and HeLa genomic DNA solutions and 0.6-160 nM of fomivirsen in mouse serum in combination with an extraction method based on alkalinization and neutralization. This method could be useful for not only detecting fomivirsen but also other functional oligonucleotides composed of phosphorothioate oligonucleotides. In summary, this study presents a practical and effective approach to the detection of the nucleic acid medicine fomivirsen.

Susceptibility of different TMEM154 genotypes in three Italian sheep breeds infected by different SRLV genotypes.

Small ruminant lentiviruses (SRLV) belong to the Retroviridae family and can cause various diseases. One of the most impacting diseases is visna-maedi, a complex disease characterized by long latencies and chronic progressive inflammatory events affecting the nervous system, lungs, mammary gland, and articular joints. A single nucleotide polymorphism (rs408593969, c.103G>A, missense mutation E35K) in the ovine transmembrane protein gene 154 (TMEM154) was identified as protective against small ruminant lentivirus infection in different herds worldwide. However, there is evidence in the scientific literature of a breed-specificity of this protective effect and, furthermore, there are still limited studies regarding the association between the animal genotype and the infecting virus genotype. Thus, the aim of this study was to further investigate the association between the animal genotype for the suggested protective mutation and the infecting virus genotype, in three different sheep breeds reared in northern Italy. The results obtained only partially confirmed the data available in the literature, as the protective effect was confirmed only for SRLV genotype A clusters, while other genotypes (namely B and E) infected AA and GA animals. Further studies with an experimental infection of specific virus genotypes in hosts with specific genotypes are required to confirm the larger number of cases the results obtained in this study.

All-in-one approaches for rapid and highly specific quantifcation of single nucleotide polymorphisms based on ligase detection reaction using molecular beacons as turn-on probes.

Rapid, simple, specific and sensitive approaches for single nucleotide polymorphisms (SNPs) detection are essential for clinical diagnosis. In this study, all-in-one approaches, consisting of the whole detection process including ligase detection reaction (LDR) and real time quantitative polymerase chain reaction performed in one PCR tube by a one-step operation on a real-time PCR system using molecular beacon (MB) as turn-on probe, were developed for rapid, simple, specific and sensitive quantifcation of SNPs. High specificity of the all-in-one approach was achieved by using the LDR, which employs a thermostable and single-base discerning Hifi Taq DNA ligase to ligate adjacently hybridized LDR-specific probes. In addition, a highly specific probe, MB, was used to detect the products of all-in-one approach, which doubly enhances the specificity of the all-in-one approach. The linear dynamic range and high sensitivity of mutant DNA (MutDNA) and wild-type DNA (WtDNA) all-in-one approaches for the detection of MutDNA and WtDNA were studied in vitro, with a broad linear dynamic range of 0.1 fM to 1 pM and detection limits of 65.3 aM and 31.2 aM, respectively. In addition, the MutDNA and WtDNA all-in-one approaches were able to accurately detect allele frequency changes as low as 0.1%. In particular, the epidermal growth factor receptor T790M MutDNA frequency in the tissue of five patients with non-small cell lung cancer detected by all-in-one approaches were in agreement with clinical detection results, indicating the excellent practicability of the developed approaches for the quantification of SNPs in real samples. In summary, the developed all-in-one approaches exhibited promising potential for further applications in clinical diagnosis.

Prediction of blood-based biomarkers and subsequent design of bisulfite PCR-LDR-qPCR assay for breast cancer detection.

BACKGROUND: Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR. METHODS: In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood. RESULTS: Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27). CONCLUSION: This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.

Single-molecule detection of cancer mutations using a novel PCR-LDR-qPCR assay.

Detection of low-abundance mutations in cell-free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR-LDR-qPCR assay capable of detecting point mutations at a single-molecule resolution in the presence of an excess of wild-type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer-binding regions as well as wild-type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single-well version of the assay detected 2-5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild-type human genomic DNA (hgDNA) from buffy coat. A 12-well (pixel) version of the assay was capable of single-molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000-2,250 GE of wild-type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.

Development of a Polymerase Chain Reaction/Ligase Detection Reaction Assay for Detection of CYP2C19 Polymorphisms.

AIMS: Cytochrome P450 2C19 (CYP2C19) genotypes are associated with differential drug metabolism. The aim of this study was to establish a reliable assay for CYP2C19 genotyping based on a polymerase chain reaction/ligase detection reaction (PCR-LDR). MATERIALS AND METHODS: Specific primers and probes were designed to detect CYP2C19*1, *2, *3, and *17. A control for each allele was prepared and used for performance evaluation. A total of 200 clinical samples were analyzed using the PCR-LDR assay and Sanger sequencing. RESULTS: The detection limit of the PCR-LDR assay was 2 ng/µL of genomic DNA. Common interfering substances in the blood did not affect the results of the detection. For the clinical samples, the results of the PCR-LDR and the Sanger sequencing were identical. Among the 200 patients, 104 (52%) were wild type (*1/*1), 64 (32%) were *1/*2, 16 (8%) were *1/*3, 8 (4%) were *2/*2, 7 (3.5%) were *2/*3, and 1 (0.5%) was *1/*7. No *3/*3 genotype was detected in these patients. CONCLUSION: This PCR-LDR assay is reliable for the detection of CYP2C19 genotypes in a clinical setting. It will be a useful tool to screen for CYP2C19 loss-of-function alleles in patients before clopidogrel and proton pump inhibitor treatment.

Detection of SNPs of T2DM susceptibility genes by a ligase detection reaction-fluorescent nanosphere technique.

OBJECTIVE: To establish a high throughput, low cost, and simple nanotechnology-based method for the detection of single nucleotide polymorphism (SNP) loci in type 2 diabetes mellitus (T2DM). METHODS: Multiplex ligase detection reaction (LDR) amplification was performed using fluorescently labeled magnetic nanosphere-bound upstream LDR probes and downstream probes labeled with a unique fluorescent group for each SNP locus. The amplified LDR products were separated by magnetic nanospheres and then scanned by fluorescence spectroscopy. Four SNP loci associated with T2DM were detected, including the rs13866634 locus in SLC30A8, rs10811661in CDKN2A/2B, rs1111875 in the HHEX gene, and rs7903146 in the TCF7L2 gene. The SNP genotype was also determined by DNA sequencing as a control. RESULTS: The SNP genotypes of the four gene loci determined by the nanosphere-based multiplex LDR method were consistent with the DNA sequencing results. The accuracy rate was 100%. CONCLUSION: A method based on multiplex PCR and LDR was established for simultaneous detection of four SNP loci of T2DM susceptibility genes.

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

The T Allele of rs8075977 in the 5'-Flanking Region of the PEDF Gene Is Associated with Reduced Risk of Coronary Artery Disease in Elderly Chinese Men.

Coronary artery disease (CAD) is a multifactorial disease with a genetic component. Pigment epithelium-derived factor (PEDF) exerts anti-inflammatory, anti-oxidant, anti-thrombotic, and anti-angiogenic effects and thus has received increasing attention as a sensitive biomarker of atherosclerosis and CAD. To explore the potential association between PEDF single nucleotide polymorphisms (SNPs) and CAD, we performed this case-control study of consecutive elderly Chinese Han male patients (n = 416) and age-matched male controls (n = 528) without a history of CAD or electrocardiographic signs of CAD. The enrolled CAD patients (age ≥ 60 years) are not biologically related. A tag approach was used to examine 100% of common variations in the PEDF gene (r2 ≥ 0.8, minor allele frequency > 0.1). PEDF tag SNPs (tSNPs) were selected using the HapMap Data-CHB which describes the common patterns of human DNA sequence variation and Tagger program. SNPs were genotyped using ligase detection reaction (LDR). Seven tSNPs (rs8075977, rs11658342, rs1136287, rs12603825, rs12453107, rs6828 and rs11078634) were selected. Among them, only one SNP, rs8075977 (C/T) located in the 5'-flanking region, showed the significant effect on the susceptibility to CAD. The frequency of its T allele was significantly higher in the controls (52.7%) than that in the CAD group (46.2%) (adjusted OR = 0.88, 95% CI: 0.80-0.96; P = 0.005). In conclusion, the T allele of rs8075977 in the 5'-flanking region of the PEDF gene may be protective for CAD. Conversely, the C allele at this variation site is associated with CAD in elderly Chinese Han men.

Highly multiplex and sensitive SNP genotyping method using a three-color fluorescence-labeled ligase detection reaction coupled with conformation-sensitive CE.

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost-effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation-dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error-prone hybridization-based detection, the multiplex assay process is complicated because of the size-based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high-resolution CE-SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar-sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three-color fluorescence-labeled probes.

Study on SNP Genotyping of Degraded DNA by Fluorescence-labeled Multiplex LDR-PCR Amplification / 中国医科大学学报

Objective In this study,a multiplex PCR amplification system was constructed based on fluorescent labeling PCR and LDR,to provide a new strategy for analyzing severely degraded DNA.Methods Eight SNP loci (rs10802248,rs10516197,rs10488372,rs2278945,rs4757318,rs4887255,rs4889002,and rs9304473) were selected.Their LDR probes and PCR primers of linked products were designed and synthesized.Ligase detection reaction,PCR amplification,and capillary gel electrophoresis (CEG) were performed to establish the multiplex LDR-PCR amplification system.Results The genotypes of these 8 loci were obtained simultaneously by the fluorescence-labeled multiplex LDR-PCR amplification method.The loci profiles obtained by fluorescence-labeled multiplex LDR-PCR amplification were in accordance with those obtained by direct sequencing of the polymorphic regions in samples from all individuals.By fluorescence-labeled multiplex LDR-PCR amplification,the 8 SNP loci were efficiently amplified from the severely degraded FFPET DNA.Conclusion Eight SNP loci results could be obtained simultaneously by using the multiplex LDR-PCR amplification system,which is a simple,efficient,and practical SNP genotyping method with accurate and reliable results for highly degraded samples.

Single nucleotide polymorphism of hsa-miR-124a affects risk and prognosis of osteosarcoma.

OBJECTIVE: To study the correlation between single nucleotide polymorphism (SNP) of hsa-miR-124a and risk and prognosis of osteosarcoma (OS). METHODS: OS patients (n = 174) hospitalized at The Second Affiliated Hospital of Harbin Medical University from January 2010 to March 2012 were selected as case group by inclusion and exclusion criteria, and healthy people (n = 150) receiving physical examination at the same duration were recruited as control group. Polymerase chain reaction-ligase detection reaction (PCR-LDR) was performed for genotyping of hsa-miR-124a rs531564. RESULTS: There were significant differences in the frequency distribution of genotypes and alleles of hsa-miR-124a rs531564 in the case and control group (all P < 0.05); the individuals carrying with CG + GG genotype showed significantly decreased risk for OS. The clinical pathological characteristics were significantly different in the patients with CC genotype and CG + GG genotype, including tumor size, tumor differentiation grading, Enneking staging, operation manner, time of chemotherapy and metastasis (all P < 0.05). The 5-year survival rate of the cases with CC genotype was significantly lower than that of the ones with CG + GG genotype (P < 0.05). CG + GG genotype, Enneking staging and operation manner were independent risk factors for prognosis of OS (all P < 0.05). CONCLUSIONS: CG +$ GG genotype of hsa-miR-124a rs531564 had decreased risk for OS and affected prognosis of OS.

Rapid detection of beta-thalassemia by LDR-ULP combined with real-time PCR / 中华检验医学杂志

Objective To establish a new method for rapid detection of β-thalassemia by investigating six clinical common mutation types.Methods Fifty cases of clinical wild-type samples and 42 cases ofβ-thalassemia samples were collected, and β-globin gene was amplified by PCR.Uniform ligation probe ( ULP) specific probes were designed for hybridization reaction to increase the reaction specificity and real-time PCR was performed to increase the sensitivity.After that, PCR products were verified by agarose electrophoresis.After examining the specificity and sensitivity, Kappa test between LDR-ULP method and reverse dot blot( RDB) method was conducted.Results Hybridization efficiency was improved 2.53 times by LDR-ULP hybridization.Each mutant type showed a significant amplification curve, whereas the wild-type had no significant curve within 40 cycles.The limit of determination of this method was 5 pg.The results of 92 cases of peripheral blood samples detected by the method of LDR-ULP and RDB were completely consistent.Conclusion In this study, a simple, inexpensive, rapid new method to detect β-thalassemia were established.

A long downstream probe-based platform for multiplex target capture.

A simple and rapid detection platform was established for multiplex target capture through generating single-strand long downstream probe (ssLDP), which was integrated with the ligase detection reaction (LDR) method for the purpose of multiplicity and high specificity. To increase sensitivity, the ladder-like polymerase chain reaction (PCR) amplicons were generated by using universal primers that complement ligated products. Each of the amplicons contained a stuffer sequence with a defined yet variable length. Thus, the length of the amplicon is an index of the specific suppressor, allowing its identification via electrophoresis. The multiplexed diagnostic platform was optimized using standard plasmids and validated by using potato virus suppressors as a detection model. This technique can detect down to 1.2 × 10(3) copies for single or two mixed target plasmids. When compared with microarray results, the electrophoresis showed 98.73-100% concordance rates for the seven suppressors in the 79 field samples. This strategy could be applied to detect a large number of targets in field and clinical surveillance.

Discriminative detection of low-abundance point mutations using a PCR/ligase detection reaction/capillary gel electrophoresis method and fluorescence dual-channel monitoring.

We applied a facile LIF dual-channel monitoring system recently developed and reported by our group to the polymerase chain reaction/ligase detection reaction/CGE method for detecting low-abundance point mutations present in a wild-type sequence-dominated population. Mutation discrimination limits and signaling fidelity of the analytical system were evaluated using three mutant variations in codon 12 of the K-ras oncogene that have high diagnostic value for colorectal cancer. We demonstrated the high sensitivity of the present method by detecting rare mutations present among an excess of wild-type alleles (one mutation among ~100 normal sequences). This method also simultaneously interrogated the allelic compositions of the test samples with high specificity through spectral discrimination of the dye-tagged ligase detection reaction products using the dual-channel monitoring system.

Association of Kallikrein gene polymorphisms with sporadic intracranial aneurysms in the Chinese population.

OBJECT: Variants of Kallikreins have been shown to be risk factors for intracranial aneurysm (IA) in a Finnish population. In the present study, the authors investigated the correlation between polymorphisms in the Kallikrein gene cluster and IAs in the Chinese population. METHODS: The association of Kallikrein variants (rs1722561 and rs1701946) with sporadic IAs was tested in 308 cases and 443 controls. The differences in allelic frequencies between patients and the control group were evaluated with the chi-square test. RESULTS: The C allele of rs1722561 showed a significant reduction in the risk of sporadic IA (OR 0.71, 95% CI 0.53-0.95; p = 0.023). However, no association of the variant rs1701946 with sporadic IA was found (OR 0.78, 95% CI 0.57-1.06; p = 0.115). CONCLUSIONS: The variant rs1722561 of Kallikreins might reduce the risk of sporadic IAs among individuals of Chinese Han ethnicity. This study confirms the association between Kallikreins and IAs.

Association of the rs11196218 polymorphism in TCF7L2 with type 2 diabetes mellitus in Asian population.

OBJECTIVE: To clarify the association of rs11196218 polymorphism in transcription factor 7-like 2 (TCF7L2) and type 2 diabetes mellitus (T2DM) in Asian population by a case-control study and meta-analysis. METHODS: In the case-control study, 1842 patients with T2DM and 7777 normal glucose-tolerant controls in the Henan province of China were genotyped for rs11196218 in TCF7L2 by PCR-ligase detection reaction. We used allele, co-dominant, dominant and recessive models to evaluate the risk association and performed a meta-analysis of the results of different genetic models in previous studies and the current study. RESULTS: The AG genotype of rs11196218 was associated with risk of T2DM in the Henan population (odds ratio 1.37, 95% confidence interval 1.06-1.78), and dominant model showed marginal significant association (1.28, 0.99-1.67). Meta-analysis of 10 studies revealed the dominant model associated with T2DM in the overall population (1.20, 1.05-1.36). When stratified by region (southern and northern China and Japan), both the AG genotype and the dominant model were associated with risk of T2DM in southern Chinese (1.31, 1.03-1.66; 1.27, 1.01-1.60, respectively). CONCLUSION: The rs11196218 polymorphism in TCF7L2 is associated with risk of T2DM in Asian population.

Three common functional polymorphisms in microRNA encoding genes in the susceptibility to hepatocellular carcinoma: a systematic review and meta-analysis.

Emerging evidences have shown that common genetic polymorphisms in microRNAs may be associated with the development of hepatocellular carcinoma (HCC); but individually published studies and previous meta-analyses revealed inconclusive results. The aims of this review and meta-analysis are to assess whether common single-nucleotide polymorphisms (SNPs) in the genes encoding the microRNAs are associated with susceptibility to HCC development and clinicopathologic characteristics of hepatitis B virus (HBV) related HCC. A computerized search was performed in PubMed, Embase, Web of Science and China BioMedicine (CBM) databases to identify relevant articles published before January 1st 2013. Ten case-control studies were assessed with a total of 3437 cases and 3437 healthy controls. Three common functional SNPs in miRNA-encoding genes were found, including miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913) and miR-499 T>C (rs3746444). This meta-analysis revealed that the miR-146a C variant was associated with a decrease in HCC risk, especially among Asian and male populations; while the miR-196a-2 T variant was associated with susceptibility to HCC among Caucasian populations. However, we failed to find any significant correlations between the miR-499 C polymorphism and HCC risks. When further stratification on HBV status was conducted, a similar trend of association between the three SNPs and the HBV-related HCC risks was observed, but these results were not statistically significant due to small sample sizes. The current meta-analysis demonstrates that SNPs contained in the genes encoding miR-146a and miR-196a-2 may play a major role in genetic susceptibility to HCC.

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR.

AIM: To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS: Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium, Lactobacillus) and three conditionally pathogenic bacteria (Enterococcus, Enterobacterium and Eubacterium) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio of the fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS: cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION: The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.