Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its arrayfor the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-l-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA of p53 gene, N-ras gene and Rb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.

Distortion-free PCA on sample space for highly variable gene detection from single-cell RNA-seq data

Single-cell RNA-seq (scRNA-seq) allows the analysis of gene expression in each cell, which enables the detection of highly variable genes (HVG) that contribute to cell-to-cell variation within a homogeneous cell population. HVG detection is necessary for clustering analysis to improve the clustering result. scRNA-seq includes some genes that are expressed with a certain probability in all cells which make the cells indistinguishable. These genes are referred to as background noise. To remove the background noise and select the informative genes for clustering analysis, in this paper, we propose an effective HVG detection method based on principal component analysis (PCA). The proposed method utilizes PCA to evaluate the genes (features) on the sample space. The distortion-free principal components are selected to calculate the distance from the origin to gene as the weight of each gene. The genes that have the greatest distances to the origin are selected for clustering analysis. Experimental results on both synthetic and gene expression datasets show that the proposed method not only removes the background noise to select the informative genes for clustering analysis, but also outperforms the existing HVG detection methods.

Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay

When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.

Establishment and evaluation based of a RIG-G gene detection system by TaqMan-MGB probe real-time PCR

Objective To establish a Taq Man-MGB fluorescent probe characterized real-time polymerase chain reaction(q PCR)method for detecting retinoic acid induced genes G(RIG-G)in human acute promyelocytic leukemia(M3).Analyze RIG-G expression levels in peripheral blood of both normal persons and M3 patients and

Rapid Detection of rpoB Gene Mutations in Rif-resistant M.tuberculosis Isolates by Oligonucleotide Microarray

Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray Results Of the 102 rifampin-resistant strains 98 （96.1%） had mutations in the rpoB genes. Conclusion Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.

APPLICATION OF GENETIC DEAFNESS GENE CHIP FOR DETECTION OF GENE MUTATION OF DEAFNESS IN PREGNANT WOMEN

Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without hearing impairment and hearing disorders family history were selected. The informed consent was signed. Peripheral blood was taken to extract genom- ic DNA. Application of genetic deafness gene chip for detecting 9 mutational hot spot of the most common 4 Chinese deafness genes, namely GJB2 （35delG, 176del16bp, 235delC, 299delAT）, GJB3 （C538T） ,SLC26A4 （ IVS72A〉G, A2168G） and mito- chondrial DNA 12S rRNA （A1555G, C1494T） . Further genetic testing were provided to the spouses and newborns of the screened carriers. Results Peripheral blood of 430 pregnant women were detected, detection of deafness gene mutation carri- ers in 24 cases（4.2%）, including 13 cases of the GJB2 heterozygous mutation, 3 cases of SLC26A4 heterozygous mutation, 1 cases of GJB3 heterozygous mutation, and 1 case of mitochondrial 12S rRNA mutation. 18 spouses and 17 newborns took further genetic tests, and 6 newborns inherited the mutation from their mother. Conclusion The common deafness genes muta- tion has a high carrier rate in pregnant women group, 235delC and IVS7-2A〉G heterozygous mutations are common.

DETECTION OF p53 GENE MUTATION OF BRONCHOSCOPIC SAMPLIES IN THE PATIENTS SUSPECTED TO LUNG CANCER

Objective: To determine the feasibility of detecting p53 gene mutations for early diagnosis of lung cancer using the samples from bronchoscopic examination. Methods: Point mutations of the exon 5-8 of p53 gene were detected in 85 bronchoscopic samples of 35 patients suspected to be lung cancer using silver staining PCR-SSCP. Results: p53 gene mutations were founded in 10 of 35 patients(28.6%). The incidence of p53 gene mutations (14.9%) was obviously higher than the cytological positive incidence(2.9%) in samples of sputum, bronchoalveolar lavage and brush, especially for the sputum(27.7%). In the bronchoscopic biopsy specimens, the incidence of p53 gene mutations (12.5%) was lower than that of pathologic positive result (50.0%). However, in view of all the bronchoscopic samples, there was no statistically difference between cytopathologic positive results (11.8%) and the incidence of p53 gene mutations (14.1%). Although the p53 mutations were most common in the samples from the patients bronchoscopically manifested as neoplasm compared with other manifestations, there was no statistical difference. It is valuable to notice that 3 patients with p53 gene mutation merely presented as bronchial inflammation in bronchoscope. Conclusion: Results indicated that the value of detecting p53 gene mutation for the diagnosis of lung cancer using the bronchoscopic samples was more superior to cytological examination and detection of p53 gene mutations in post-bronchoscopic sputum was easy and effective, may be used as a valuable method for early diagnosis of lung cancer.

DETECTION OF GENE MUTATION IN SPUTUM OF LUNG CANCER PATIENT

Lungcancerisacommonmalignanttumor,whichhasahighincidenceandmortalityrate.Therefore,itisnecessarytoseekanewmethodforthediagnosis,especiallytheearlydiagnosisoflungcancer.Thedevelopmentofmolecularbiologymakesthegenediagnosisoflungcancerpossible.PCR-SSCP...

Detecting the polymorphism sites of p53 and Fas genes of Han population in Zhejiang province

BACKGROUND： It is of significance for single nucleotide polymorphisms （SNPs）, a difference of rank, which exists widely in biology, genetics and other fields. OBJECTIVE： To detect polymorphism sites in exon-4 of p53 gene, promotor of Fas gene and intron-7 of Fas gene of healthy people in Han nationality in Zhejiang province. DESIGN： Simple random sampling. SETTING： Department of Surgery of the 118 Hospital of Chinese PLA.PARTICIPANTS： A total of 80 healthy people in Han nationality were selected from hospitals in Zhejiang province from August 2005 to January 2006. There were 43 males and 37 females aged from 3 to 78 years with the mean age of 39.5 years, and all subjects were consent. DNA which was used in genetic analysis was selected from peripheral venous blood of all subjects and maintained at -20℃.METHODS： Polymorphism sites in exon-4 of p53 gene, promotor of Fas gene and intron-7 of Fas gene were detected with directly DNA sequencing technique. MAIN OUTCOME MEASURES ： Polymorphism sites in exon-4 of p53 gene, promotor of Fas gene and intron-7 of Fas gene of healthy people in Han nationality in Zhejiang province. RESULTS： A total of 80 samples were involved in the final analysis. SNPs sites were found at the 119^th base of exon-4 of p53 gene （the 72^nd codon of p53 gene）, the 670^th base of upper start codon in promotor of Fas gene （Fas-670）, and the 995^th base of intron-7 of Fas gene, especially SNPs in the 995^th base of intron-7 pf Fas gene, i.e. C→A transversion, was a new site.CONCLUSION ： One unknown SNPs site is discovered in intron-7 of Fas gene of people in Han nationality in Zhejiang province. This study also proves that the 72^nd codon exists in p53 gene and the -670 polymorphism site exists in promotor of Fas gene.

Molecular Detection and Disease Resistance Identification of Transgenic Wheat with pti5-vp16 Gene

The immature embryos of wheat cultivars Liaochun10, Tiechun1 and Fengqiang3 were bombarded with gold particles coated with pti5 vp16 by gene gun and disease resistant regenerated plants were attained. In order to confirm that the plants are genuine transformed ones, a series of molecular tests were conducted as follows. Firstly, transient GUS expression test on embryos two days after bombardment was done. There were many obvious blue spots produced on the surface of bombarded embryos after GUS staining, in which the maximum reached to 85 blue spots per embryo. Secondly, PCR test was performed with DNA from the regenerated plants obtained after double selection with ppt. 6 plants were found PCR test positive. At last, further verification analysis using dot hybridization and southern blotting was carried out on those PCR positive plants and the strong hybridization signals appeared as expected. All the above tests were uniformly indicated that the disease resistant regenerated plants were true transgenic plants. When inoculated with Blumeria graminis, transgenic wheat plants of PCR positive results were mostly resistant(R) after 7 days, and resis tant, moderate resistant(MR), moderate susceptible(MS) at 14 days respectively. The disease severity of them was distinctively lighter than that of control.

Ratio quantification of gene dosage by Agilent 2100 Bioanalyzer for detection of somatic gene deletions

We applied the Agilent 2100 Bioanalyzer, a microfluidics-based electrophoresis instrument, and introduced an accurate and consistent parameter, the relative product yield ratio (ROY), to detect somatic gene deletions in tumor cells. Briefly for such purpose, the Agilent 2100 Bioanalyzer quantified the ROY of a target gene to an endogenous internal control, both of which were initially co-amplified by Robust-Dosage PCR (RD- PCR). Herein, we extensively validated this approach. We first tested the effect of well positions on the Agilent DNA chip. We found that no matter which wells the samples were loaded in, the ROY was consistent with coefficient of variation (CV) < 2%. Then we tested the effect of product concentrations that varied 8-fold, and the ROY was also consistent with CV < 3.5%. Furthermore, we applied this approach to identify six somatic KRAS deletions in non-small cell lung cancer patients, confirming our previous findings. Thus, the Agilent 2100 Bioanalyzer is simple, accurate, quick, and ultimately able to replace conventional gel electrophoresis for the detection of somatic gene deletions.

Transfer and Detection of barstar Gene to Maize Inbred Line 18-599 (White) by Particle Bombardment

In China, the purity of maize hybrid strain is discomforting to the development of seed industrialization. Finding a new method for reproduction of maize hybrid strain is necessary. In this study, using particle bombardment, barstar gene was transferred into maize inbred line 18-599 （White）, which is an antiviral and high quality maize inbred line. By molecular detection of the anther of transgenic maize, two plants transferred with barstar gene were gained in this study, which are two restorer lines. The two plants showed normal male spike, and lively microspores. But the capacity of the two restorer lines should be studied in the future. The aim of this study is to find a new method of reproduction of maize hybrid strain using engineering restorer lines and engineering sterility lines by gene engineering technology.

GENE ENGINEERING EB VIRUS MEMBRANE ANTIGEN IN DETECTION OF MA-IgA ANTIBODY(COMPARISON WITH VCA-IgA AND EA-IgA ANTIBODIES)

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluo-rescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pretreatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.

Prediction of Lung Cancer Stage Using Tumor Gene Expression Data

Lung cancer remains a significant global health challenge and identifying lung cancer at an early stage is essential for enhancing patient outcomes. The study focuses on developing and optimizing gene expression-based models for classifying cancer types using machine learning techniques. By applying Log2 normalization to gene expression data and conducting Wilcoxon rank sum tests, the researchers employed various classifiers and Incremental Feature Selection (IFS) strategies. The study culminated in two optimized models using the XGBoost classifier, comprising 10 and 74 genes respectively. The 10-gene model, due to its simplicity, is proposed for easier clinical implementation, whereas the 74-gene model exhibited superior performance in terms of Specificity, AUC (Area Under the Curve), and Precision. These models were evaluated based on their sensitivity, AUC, and specificity, aiming to achieve high sensitivity and AUC while maintaining reasonable specificity.

西安地区256例Gene Xpert MTB/RIF阳性肺结核患者rpoB基因突变特征分析

目的分析西安地区256例利福平耐药实时荧光定量核酸扩增检测技术(Gene Xpert MTB/RIF)阳性肺结核患者rpoB基因突变特征。方法本研究为回顾性分析。256株结核分枝杆菌(MTB)菌株分离自2020年6月至2022年6月于陕西省结核病防治院就诊的Gene Xpert MTB/RIF阳性肺结核门诊及住院患者,菌株无重复收集,来自痰液标本174份、支气管肺泡灌洗液标本82份,患者年龄(45.67±8.36)岁。采用DNA直接测序法对256株利福平耐药MTB菌株rpoB基因的PCR产物进行分析。将256株利福平耐药MTB菌株根据利福平耐药程度分为低、中、高耐药MTB菌株,采用χ^(2)检验比较3种菌株突变位点。人工诱导3株利福平耐药MTB菌株,采用DNA直接测序法对其rpoB基因的PCR产物进行分析。结果测序报告显示,256株利福平耐药MTB菌株中有253株发生rpoB基因位点突变,突变率为98.83%(253/256)。突变类型包括C→T、T→G、C→G、A→T、C→A、A→G、G→A、G→T、T→C、A→C共计10种,涉及丝氨酸、亮氨酸、丙氨酸、组氨酸、酪氨酸、谷氨酸、赖氨酸、精氨酸、天冬氨酸、脯氨酸、蛋氨酸、缬氨酸、异亮氨酸、甘氨酸共14个氨基酸密码子,均为点突变。利福平耐药菌株突变主要集中在531位[53.75%(136/253)]、526位[23.32%(59/253)],其他位点包括513、516、533、515、513、532、522、511、519、518、533。高耐药MTB菌株531位氨基酸突变发生率与低、中耐药MTB菌株比较[66.91%(91/136)比37.88%(25/66)、37.04%(20/54)],差异有统计学意义(χ^(2)=22.154,P<0.001);低、中耐药MTB菌株531位氨基酸突变发生率比较,差异无统计学意义(P>0.05)。低、中、高耐药MTB菌株526位氨基酸突变发生率比较[22.73%(15/66)、25.93%(14/54)、22.06%(30/136)],差异无统计学意义(χ^(2)=0.331,P=0.847)。人工诱导的3株利福平耐药MTB菌株均发生rpoB基因位点突变,低、中耐药MTB菌株突变均位于526位点,高耐药MTB菌株突变位于531位点。结论西安地区Gene Xpert MTB/RIF阳性肺结核患者rpoB基因突变率较高,以点突变为主,主要集中在531位、526位,531位TCG→TTG突变在rpoB基因突变类型中突变频率最高,且与高耐药有关。

痰涂片镜检、基因芯片技术及GeneXpert MTB/RIF对疑似肺结核患者的检测效能分析

目的分析痰涂片镜检、基因芯片及多色巢式荧光定量聚合酶链反应(GeneXpert MTB/RIF)对疑似肺结核患者的检测效能。方法选取2017年1月至2022年12月该院收治的疑似肺结核患者97例作为研究对象,均实施痰涂片镜检、基因芯片、GeneXpert MTB/RIF及痰液罗氏培养检查。以痰液罗氏培养结果为金标准,探究不同检测方式及联合检测对疑似肺结核的诊断效能,采用Kappa值检验与金标准诊断结果的一致性。结果痰液罗氏培养检测结果显示,97例疑似肺结核患者阳性55例,阴性42例,阳性检出率为56.70%(55/97)。痰液罗氏培养检出非结核分枝杆菌18株,包括鸟分枝杆菌4株,胞内分枝杆菌9株,偶发分枝杆菌1株,堪萨斯分枝杆菌3株,海分枝杆菌1株。痰涂片镜检检出阳性34例,真阳性29例,阳性检出率为35.05%(34/97),基因芯片检出阳性41例,真阳性37例,阳性检出率为42.27%(41/97);GeneXpert MTB/RIF检出阳性44例,真阳性37例,阳性检出率为45.36%(44/97)。基因芯片、GeneXpert MTB/RIF灵敏度、准确率高于痰涂片镜检,基因芯片与GeneXpert MTB/RIF敏感度一致,但基因芯片特异度高于GeneXpert MTB/RIF。非结核分枝杆菌中,痰涂片镜检检出鸟分枝杆菌1株、胞内分枝杆菌2株,基因芯片检出胞内分枝杆菌1株,GeneXpert MTB/RIF检出鸟分枝杆菌1株。痰涂片镜检、基因芯片、GeneXpert MTB/RIF三项联合检出阳性55例,真阳性53例,三项联合检测灵敏度为96.36%(53/55)、特异度为95.24%(40/42)、准确率为95.88%(93/97),均高于单一方法检测的灵敏度与准确率,差异均有统计学意义(P<0.05)。痰涂片镜检与痰液罗氏培养一致性为68.04%(Kappa=0.59);基因芯片与痰液罗氏培养一致性为77.32%(Kappa=0.70);GeneXpert MTB/RIF与痰液罗氏培养一致性为74.23%(Kappa=0.66);三项联合与痰液罗氏培养一致性为95.88%(Kappa=0.89)。结论较GeneXpert MTB/RIF、痰涂片镜检技术,基因芯片诊断效能及一致性更高,且3种技术联合诊断效能更高,临床可根据需求选择适宜诊断技术。

一株苯酚降解菌(Alcaligenes faecalis BC2001)的PCR检测及其苯酚降解特性研究

根据苯酚羟化酶基因高度保守序列设计一对该基因的特异引物。采用该特异引物从粪产碱菌BC2001总DNA中扩增一条大小为684 bp的片段。将该DNA片段进行序列分析,发现其与Proteobacterium L-18菌(登录号为:AY346142)的基因序列的同源性为97%。另降解试验表明BC2001菌在pH=6时(质量浓度为500mg.L-1)其生长最佳,而在苯酚质量浓度为300 mg.L-1时也是菌株具降解能力的有效例子。

Gene X-pert MTB/RIF检测对肺结核病的诊断价值研究

目的 研究分析肺结核病应用Gene X-pert MTB/RIF检测的诊断价值。方法 选取2022年3—10月在本院就诊的疑似肺结核病患者164例为研究对象,所有患者均给予结核分枝杆菌培养检查、痰涂片抗酸染色法诊断、Gene X-pert MTB/RIF检测,以结核分枝杆菌培养检查结果为金标准,比较痰涂片抗酸染色法诊断与Gene X-pert MTB/RIF检测的阳性率以及诊断准确性、灵敏度、特异度。结果 痰涂片抗酸染色法诊断阳性率为21.34%(35/164),Gene X-pert MTB/RIF检测阳性率为35.98%(59/164),Gene X-pert MTB/RIF检测明显高于痰涂片抗酸染色法诊断(P<0.05)。痰涂片抗酸染色法诊断的准确性为85.37%,灵敏度为59.65%,Gene X-pert MTB/RIF检测的准确性为95.12%,灵敏度为94.74%,Gene X-pert MTB/RIF检测明显高于痰涂片抗酸染色法诊断(P<0.05)。结论 在肺结核病诊断中,Gene X-pert MTB/RIF检测的阳性率更高,同时诊断准确率与灵敏度也更高,为肺结核病的诊断与治疗提供了可靠的指导依据。

血清SHBG联合Gene-Xpert检测对涂阴肺结核的诊断价值

目的分析血清性激素结合球蛋白(SHBG)联合Gene-Xpert检测对涂阴肺结核的诊断价值。方法选取山东省聊城市人民医院2017年6月至2019年6月88例疑似涂阴肺结核患者为研究对象,以肺泡灌洗液结核分枝杆菌培养为金标准诊断出其他肺部感染患者30例(非结核性肺部炎症组),涂阴肺结核患者58例(涂阴肺结核组)。所有患者均行胸部CT和X线片检查,对病变部位进行灌洗,收集肺泡灌洗液行Gene-Xpert检测,采用酶联免疫吸附试验法检测血清中SHBG水平。采用受试者工作特征(ROC)曲线评价血清SHBG水平对涂阴肺结核的诊断价值,以肺泡灌洗液结核分枝杆菌培养为金标准评价SHBG联合Gene-Xpert检测对涂阴肺结核的诊断价值。结果88例疑似涂阴肺结核患者中,X线片、CT检查有斑片影、云絮影者占64.77%。与非结核性肺部炎症组相比,涂阴肺结核组血清SHBG水平明显升高,差异有统计学意义(P<0.05)。肺泡灌洗液诊断涂阴肺结核的ROC曲线下面积(AUC)为0.856(95%CI:0.756~0.922),截断值为39.94 ng/mL,特异度为79.31%,灵敏度为80.00%。Gene-Xpert检测诊断涂阴肺结核的灵敏度为74.14%,特异度为73.33%,准确度为73.86%。血清SHBG联合Gene-Xpert检测诊断涂阴肺结核的灵敏度为93.33%,特异度为94.83%,准确度为94.32%。与单独血清SHBG和Gene-Xpert检测相比,联合检测的灵敏度、特异度、准确度均明显升高,差异有统计学意义(P<0.05)。结论血清SHBG联合Gene-Xpert检测可提高涂阴肺结核的诊断性能。