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.

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.

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.

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

DETECTION OF GENE MUTATION IN SPUTUM OF LUNG CANCER PATIENT

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

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.

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.

Identification and Characterization of Putative Virulent Genes in Streptococcus equi ssp. zooepidemicus

Suppression subtractive hybridization （SSH） was performed with virulent strain ATCC35246 and avirulent strain ST171 to identify novel genes associated with virulence in Streptococcus equi ssp. zooepidemicus （SEZ）. There were fourteen genomic regions that only presented in virulent strain ATCC35246. These regions encoded 14 proteins, some of them were homologous to proteins associated with cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems, and other unknown functions. Primers for 6 particular regions were designed from the already published SEZ sequence. Then, we used PCR to evaluate the distribution and conservation of these 6 DNA fragments in various SEZ strains collected from different sources, regions, groups, and times. The results showed that these 6 DNA fragments were widely distributed in SEZ strains, yet they were not existence in the avirulent strain ST171. Moreover, these fragments could not be detected in other Streptococcus groups.

Complex heterozygous mutations in hereditary spherocytosis:A case report

BACKGROUND The aim of this study was to investigate the complex heterozygous mutations of ANK1 and SPTA1 in the same individual and improve our understanding of hereditary spherocytosis(HS)in children.We also hope to promote the application of gene detection technology in children with HS,with the goals of identifying more related gene mutations,supporting the acquisition of improved molecular genetic information to further reveal the pathogenesis of HS in children,and providing important guidance for the diagnosis,treatment,and prevention of HS in children.CASE SUMMARY A 1-year and 5-month-old patient presented jaundice during the neonatal period,mild anemia 8 months later,splenic enlargement at 1 year and 5 months,and brittle red blood cell permeability.Genetic testing was performed on the patient,their parents,and sister.Swiss Model software was used to predict the protein structure of complex heterozygous mutations in ANK1 and SPTA1.Genetic testing revealed that the patient harbored a new mutation in the ANK1 gene from the father and a mutation in the SPTA1 gene from the mother.Combined with the clinical symptoms of the children,it is suggested that the newly discovered complex heterozygous mutations of ANK1 and SPTA1 may be the cause,providing important guidance for revealing the pathogenesis,diagnosis,treatment,and promotion of gene detection technology in children with HS.CONCLUSION This case involves an unreported complex heterozygous mutation of ANK1 and SPTA1,which provides a reference for exploring HS.

ACTA2 mutation is responsible for multisystemic smooth muscle dysfunction syndrome with seizures:A case report and review of literature

BACKGROUND ACTA2 gene is a specific gene that encodes actinα2.Multisystem smooth muscle dysfunction syndrome(MSMDS)is a multisystem disease characterized by aortic and cerebrovascular lesions caused by ACTA2 gene mutations.There have been many reports of cardiac,pulmonary and cerebrovascular lesions caused by MSMDS;however,few studies have focused on seizures caused by MSMDS.CASE SUMMARY Our patient was a girl aged 7 years and 8 mo with recurrent cough,asthma and seizures for 7 years.She was diagnosed with severe pneumonia,congenital heart disease,cardiac insufficiency,and malnutrition in the local hospital.Cardiac ultrasonography revealed congenital heart disease,patent ductus arteriosus(with a diameter of 0.68 cm),left coronary arteriectasis,patent oval foramen(0.12 cm),tricuspid and pulmonary regurgitation,and pulmonary hypertension.Cerebral magnetic resonance imaging and magnetic resonance angiography indicated stiffness in the brain vessels,together with multiple aberrant signaling shadows in bilateral paraventricular regions.A heterozygous mutation(c.536G>A)was identified in the ACTA2 gene,resulting in generation of p.R179H.Finally,the girl was diagnosed with MSMDS combined with epilepsy.The patient had 4 episodes of seizures before treatment,and no onset of seizure was reported after oral administration of sodium valproate for 1 year.CONCLUSION MSMDS has a variety of clinical manifestations and unique cranial imaging features.Cerebrovascular injury and white matter injury may lead to seizures.Gene detection can confirm the diagnosis and prevent missed diagnosis or misdiagnosis.

Fatal community-acquired bloodstream infection caused by Klebsiella variicola:A case report

BACKGROUND Klebsiella pneumoniae(K.pneumoniae)is an infective microorganism of worldwide concern because of its varied manifestations and life-threatening potential.Genetic analyses have revealed that subspecies of K.pneumoniae exhibit higher virulence and mortality.However,infections with Klebsiella subspecies are often misdiagnosed and underestimated in the clinic because of difficulties in distinguishing K.pneumoniae from its subspecies using routine tests.This case study reports the rapid and fatal effects of K.pneumoniae subspecies.CASE SUMMARY A 52-year-old male patient was febrile and admitted to hospital.Examinations excluded viral and fungal causes along with mycoplasma/chlamydia and parasitic infections.Bacterial cultures revealed blood-borne K.pneumoniae sensitive to carbapenem antibiotics,although corresponding treatment failed to improve the patient’s symptoms.His condition worsened and death occurred within 72 h of symptom onset from sepsis shock.Application of the PMseq-DNA Pro high throughput gene detection assay was implemented with results obtained after death showing a mixed infection of K.pneumoniae and Klebsiella variicola(K.variicola).Clinical evidence suggested that K.variicola rather than K.pneumoniae contributed to the patient’s poor prognosis.CONCLUSION This is the first case report to show patient death from Klebsiella subspecies infection within a short period of time.This case provides a timely reminder of the clinical hazards posed by Klebsiella subspecies and highlights the limitations of classical laboratory methods in guiding anti-infective therapies for complex cases.Moreover,this report serves as reference for physicians diagnosing similar diseases and provides a recommendation to employ early genetic detection to aid patient diagnosis and management.

Comparison of Hematological Parameters and Critical Value Analysis in the Prevention and Control of Thalassaemia in Hainan Li and Han Nationalities

Objective: To compare the distribution of “mean corpuscular hemoglobin”-MCV, “mean corpuscular volume”-MCH, “hemoglobin”-HGB, “hemoglobin A”-HbA and “hemoglobin A2”-HbA2 in α and β thalassemia hematology screening between Li and Han nationality, and analyze the best diagnostic cut-off value. Methods: Select 7816 middle school students from Li nationality area as the research object, collect peripheral blood for blood cell analysis, hemoglobin electrophoresis and thalassaemia gene detection, and compare the difference in hematological parameters of common thalassemia genotype between Li and Han nationalities. Taking the genetic test results as the gold standard, construct the receiver operator characteristic curve (ROC curve) of relevant hematology parameters, calculate the Youden index and take its maximum diagnostic cut-off point as the best critical value. Results: Comparison of hematological parameters of common thalassemia genotypes showed that the average value of MCH and MCV of -α3.7/-α4.2 type in Li nationality was lower than that of Han nationality, and the average value of HbA2 of CD41-42/βN type was higher than that of Han nationality, there was no significant difference among other genotypes. ROC curve analysis shows that the MCH, MCV, and HGB values have poor diagnostic efficiency for thalassaemia, HbA has a slightly better diagnostic efficiency for α thalassaemia, and the optimal cut-off values of HbA for Li and Han nationalities are 96.95% and 97.75%, respectively;HbA2 has better screening efficiency for β-thalassemia, and the optimal cut-off values of HbA2 for Li and Han nationalities are 4.20% and 3.45% respectively. Conclusion: In the prevention and control screening of thalassaemia in the Li and Han nationalities, hemoglobin electrophoresis technology has a better diagnostic efficiency.

The diagnosis and treatment process of one clinical case of juvenile glycogen storage disease in current medical model in china

Glycogen storage disease type Ⅱ, also known as Pompe disease (PD), is a kind of congenital metabolic myopathy, the cause of this disease is the barrier of glycogen disintegration due to the shortage of acid alpha-1,4-glucosidase enzyme. The prevalence of PD ranges between 1:40,000 and 1:300,000 and is dependent on ethnic and geographical factors. The main clinical manifestation of this myopathy is the injury of muscle organization. According to the difference in age and developmental speed, we can divide this disease into three types: the infantile type, the juvenile type and the adult type, and the latter two types are called the late onset type. Because the symptom of the late onset form is not typical, the mutual mixture of the late onset type and some chronic myopathy always takes place, which can even cause evade diagnosis and misdiagnosis, thus it is supposed to be highly focused on. At present, there is a shortage of effective therapeutic methods to cope with this myopathy all over the world. The following part is a report about the clinical data and the rehabilitation intervention of a patient who caught the juvenile type of GSD-Ⅱ, which is combined to home and abroad literatures, the purpose of this report is to help enhance clinical physicians’ cognition of this disease in the future therapy. At the same time, we combine with the current medical mode and system in China to reflect on the prevention and treatment of such diseases.

DETECTION OF GENES FOR HEAT-STABLE ENTEROTOXIN IN ESCHERICHIA COLI BY BIOTINYLATED ST-DNA PROBES

Reference strains of enterotoxigenic Escherichia coli (ETEC), non-enterotoxigenic Escherichia coli (non-ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), and other enteropathogenic bacteria were used to prove the reliability of BIO-ST-DNA probe hybridization. In addition, 417 strains of E. coli isolated from children with diarrheal diseases in Shanxi Children's Hospital were examined for BIO-ST-DNA probe hybridization. In the test, BIO-ST-DNA hybridization was compared with suckling mouse assay in identifying ST-ETEC. The results obtained by both methods showed no significant difference. It was found that identification of ST-ETEC using hybridization is a simple, sensitive and more practical method.

Detection of p53 gene mutations in skeletal muscle cell of patients with chronic heart failure

Progressive deterioration of physical work capacity in congestive heart failure (CHF) is often attributed to ongoing skeletal muscle atrophy and abnormalities in muscle metabolism. The purpose of the present study was to investigate if mutations in the p53 gene thought to be a promotor of apoptosis are involved in intrinsic apoptotic abnormalities in skeletal muscle of patients (pts) with CHF. Percutaneous needle biopsy from the m. vastus lateralis were obtained from 19 pts with CHF (LV EF 25%±10%). Single strand confirmation polymorphism analysis of polymerase chain reation products (PCR SSCP analysis) was used for detection of mutations in exon 5 8 of the p53 gene in skeletal Heart Center, University Leipzig, Germany (Yu JT, Adams V, Fiehn E, Schuler G and Hambrecht R) Institut of Pathology, University Freiburg, Germany (Ye J and Riede U)muscle cells. Four of 19 muscle specimens (21%) showed mobility shifts. To characterize the nuleotide sequence alterations specimens were examined further by direct sequence analysis of PCR product. Two of four specimens showing a band shift in the SSCP analysis exhibited a mutated p53 sequence. Sequence analysis revealed that these alteratons were point mutation exon 8 (14482, G→A) and deletion in exon 5 (13143 13157). A high frequency of p53 mutations was detected in skeletal muscle cells of patients with chronic heart failure. These findings suggest a role for apoptosis in the progression of intrinsic skeletal muscle abnormalities and consequently of exercise intolerance in chronic heart failure.

PacBio Sequencing and Its Applications

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation （SV） in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with dis- eases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Addition- ally, PacBio＇s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.