1.Direct Detection of Mycobacterium Tuberculosis in Respiratory Specimens by Ligase Chain Reaction.
Korean Journal of Clinical Pathology 1998;18(2):215-219
BACKGROUND: The most common clinical manifestation of tuberculosis is respiratory tract infections. Currently, respiratory tract tuberculosis is diagnosed by using X-ray, acid-fast smear, culture, or DNA probe technology. The nucleic acid amplification technologies include the polymerase chain reaction (PCR) and the ligase chain reaction (LCR). The potential utility of LCx (Abbott Lab.) kit for the detection and identification of Mycobacterium tuberculosis in respiratory specimens has been measured. METHODS: Four different methods such as acid-fast smear, culture, PCR, and LCR were evaluated using 58 specimens isolated from patients. The IS6110 sequences for Mycobacterium tuberculosis synthesized and provided by Applied Biosystems were used for PCR procedure. The LCR assay using LCx kit was performed according to the manufacturer's instruction (Abbott Lab., U.S.A.). RESULTS: Sensitivity, specificity, and positive and negative predicative values for acid-fast smear method were 72, 100, 100 and 89%, respectively and were 89, 100, 100 and 95%, respectively for culture method. Whereas those values for PCR method were 78, 100, 100, and 91% respectively, and those for LCR were 100, 95, 90 and 100%, respectively. CONCLUSIONS: The LCR assay performed on respiratory specimens for the detection of Mycobacterium tuberculosis has been evaluated as a highly effective method among 4 different identification systems.
DNA
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Humans
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Ligase Chain Reaction*
;
Mycobacterium tuberculosis*
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Mycobacterium*
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Polymerase Chain Reaction
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Respiratory System
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Respiratory Tract Infections
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Sensitivity and Specificity
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Tuberculosis
2.Detection of the genetic abnormalities in patients with mental retardation using multiplex ligation-dependent probe amplification assay.
Lina ZHU ; Chunzhi WANG ; Xiao YANG ; Yan WANG ; Xin LIU ; Xiyu HE
Chinese Journal of Medical Genetics 2009;26(6):644-647
OBJECTIVETo investigate the relationship between subtelomeric rearrangements and idiopathic mental retardation (MR).
METHODSThirty unrelated patients were recruited using strict selection criteria. Patients were screened by multiplex ligation-dependent probe amplification (MLPA) for subtelomeric imbalance.
RESULTSFive subtelomeric deletions/duplications were identified. They were: 4p deletion, 21p duplication, 10p duplication combined with 4p deletion, 15p duplication, and 9p deletion combined with 3p duplication. These subtelomeric rearrangements were previously unidentified by conventional technique.
CONCLUSIONChildren with unexplained mental retardation are related with subtelomeric rearrangements. MLPA is a rapid and an effective technique for detecting genetic abnormalities in patients with idiopathic MR.
Child ; Chromosome Aberrations ; Female ; Gene Deletion ; Gene Duplication ; Humans ; Intellectual Disability ; diagnosis ; genetics ; Ligase Chain Reaction ; methods ; Male
3.Detection of microdeletion in Williams syndrome by multiplex ligation-dependent probe amplification.
Yuan-yuan PENG ; Yan MENG ; Zheng-qing QIU ; Ou WANG ; Shang-zhi HUANG
Chinese Journal of Medical Genetics 2009;26(4):369-373
OBJECTIVETo establish a method of multiplex ligation-dependent probe amplification (MLPA) for clinical screening of Williams syndrome (WS) and for routine use in WS diagnosis.
METHODSProbes for MLPA were designed according to the frequent deletion regions, and used to screen the two patients suspected with Williams syndrome, and the density of the bands were analyzed with software. Linkage analysis using polymorphic markers was performed to confirm the positive result of MLPA.
RESULTSThe MLPA data indicated that the two children had possible microdeletions in the WS critical region. The deletions were confirmed and both were maternal origin by polymorphism analysis.
CONCLUSIONMLPA is a quick and convenient method for detecting deletion or duplication mutations. It can provide reliable and helpful information for clinical diagnose of Williams syndrome.
Child ; Humans ; Ligase Chain Reaction ; methods ; Male ; Oligonucleotide Probes ; genetics ; Sequence Deletion ; Williams Syndrome ; diagnosis ; genetics ; Young Adult
4.Development of a gap ligase chain reaction for detection of Chlamydia trachomatis in newborn infants.
Hong WEI ; Shi-xiao WU ; Jia-lin YU ; Jun YANG ; Guan-xin LIU
Chinese Journal of Pediatrics 2003;41(8):578-581
OBJECTIVETo establish a gap ligase chain reaction (G-LCR) assay for the detection of Chlamydia trachomatis (Ct) in neonates with pneumonia.
METHODSA G-LCR DNA amplification assay that targeted the outer major membrane protein gene (omp1) of Ct was developed to detect Ct. The sensitivity and specificity of the G-LCR test was examined by the use of highly purified elementary bodies (EBs). Nasopharyngeal swabs taken from 328 neonates with pneumonia were analyzed by Gap-LCR and cell culture.
RESULTSThe detection limit of G-LCR was 2 EBs. G-LCR could detect five species of Ct and was not cross-reacted with C psittaci and other bacteria. The prevalence of Ct in 328 neonates with pneumonia, using an expanded gold standard of a positive cell culture or two confirmed positive non-culture tests, was 21% (69/328). After analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive values for the G-LCR were 98.6%, 100%, 100% and 99.6%, respectively; whereas those for culture were 86.9%, 100%, 100% and 96.6%, respectively.
CONCLUSIONThis study demonstrated that the G-LCR was a highly sensitive nonculture technique and good alternative test for the detection of chlamydial infections.
Chlamydia Infections ; diagnosis ; microbiology ; Chlamydia trachomatis ; genetics ; Female ; Humans ; Infant, Newborn ; Ligase Chain Reaction ; methods ; Male ; Sensitivity and Specificity
5.Establishment of the multiplex quantitative ligase chain reaction for detecting mutations of deafness genes.
Chinese Journal of Medical Genetics 2010;27(5):530-534
OBJECTIVETo establish a low-cost, convenient and accurate multiplex quantitative ligase chain reaction (MQ-LCR) technique to detect the five common mutations in Chinese patients with deafness.
METHODSPrimers and probes for 5 common mutations of deafness genes, i.e., GJB2 gene 235delC and 299-300delAT, mtDNA A1555G, SLC26A4 gene IVS7-2 A>G and 2168A>G, were designed and synthesized. The technique for those mutations was established, and the reliability of the technique was tested in 98 patients with impaired hearing and 30 children with normal hearing, who were randomly selected from the ENT in Children's Hospital of Fudan University. The subjects were detected by MQ-LCR and direct DNA sequencing of PCR products, following a double-blind approach. Finally the results from the two methods were compared.
RESULTSThe results revealed 48 cases carried two mutations, 31 cases carried heterozygous mutations in the 98 deaf children, and 3 had heterozygous mutation in 30 normal controls. These results were consistent with that from DNA sequencing. No false positive and false negative result was obtained.
CONCLUSIONThe MQ-LCR technique established in this study is of low-cost, convenience, accuracy, high sensitivity and high specificity. It is suitable for large-scale detection and preventive diagnosis of mutations in deafness.
Base Sequence ; Case-Control Studies ; Child ; Child, Preschool ; Connexins ; Deafness ; diagnosis ; genetics ; Female ; Humans ; Infant ; Ligase Chain Reaction ; methods ; Male ; Molecular Sequence Data ; Mutation
6.Genetic Analysis of Dystrophin Gene for Affected Male and Female Carriers with Duchenne/Becker Muscular Dystrophy in Korea.
Bo Lyun LEE ; Sook Hyun NAM ; Jun Hwa LEE ; Chang Seok KI ; Munhyang LEE ; Jeehun LEE
Journal of Korean Medical Science 2012;27(3):274-280
Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direct DNA sequencing for some patients who had negative results using the above methods. The overall mutation detection rate was 72.4% (21/29) in DMD/BMD patients, identifying deletions in 58.6% (17/29). Most of the deletions were confined to the central hot spot region between exons 44 and 55 (52.9%, 7/19). The percentage of deletions and duplications revealed by MLPA was 45.5% (5/11) and 27.2% (3/11), respectively. Using the MLPA method, we detected mutations confirming their carrier status in all female relatives and symptomatic female patients. In one patient in whom MLPA revealed a single exon deletion of the dystrophin gene, subsequent DNA sequencing analysis identified a novel nonsense mutation (c.4558G > T; Gln1520X). The MLPA assay is a useful quantitative method for detecting mutation in asymptomatic or symptomatic carriers as well as DMD/BMD patients.
Adolescent
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Adult
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Child
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Child, Preschool
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DNA Mutational Analysis
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Dystrophin/*genetics
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Exons
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Female
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Heterozygote
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Humans
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Infant
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Ligase Chain Reaction
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Male
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Multiplex Polymerase Chain Reaction
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Muscular Dystrophy, Duchenne/*genetics
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Mutagenesis, Insertional
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Republic of Korea
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Sequence Analysis, DNA
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Sequence Deletion
7.Implication of leucyl-tRNA synthetase 1 (LARS1) over-expression in growth and migration of lung cancer cells detected by siRNA targeted knock-down analysis.
Seung Hun SHIN ; Ho Shik KIM ; Seung Hyun JUNG ; Hai Dong XU ; Yong Bok JEONG ; Yeun Jun CHUNG
Experimental & Molecular Medicine 2008;40(2):229-236
Molecular mechanism of lung carcinogenesis and its aggressive nature is still largely elusive. To uncover the biomarkers related with tumorigenesis and behavior of lung cancer, we screened novel differentially expressed genes (DEG) in A549 lung cancer cell line by comparison with CCD-25Lu, normal pulmonary epithelial cell line, using annealing control primer(ACP)- based GeneFishing system. Of the DEGs, over-expression of leucyl-tRNA synthetase 1 (LARS1) was prominent and this up-regulation was confirmed by immunoblotting and real-time quantitative RT-PCR analysis. In addition to A549 cell line, primary lung cancer tissues also expressed higher level of LARS1 mRNA than their normal counter tissues. To explore the oncogenic potential of LARS1 over-expression in lung cancer, we knocked-down LARS1 by treating siRNA and observed the tumor behavior. LARS1 knock-down cells showed reduced ability to migrate through transwell membrane and to form colonies in both soft agar and culture plate. Taken together, these findings suggest that LARS1 may play roles in migration and growth of lung cancer cells, which suggest its potential implication in lung tumorigenesis.
Base Sequence
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Blotting, Western
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Cell Line, Tumor
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*Cell Movement
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DNA Primers
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Humans
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Leucine-tRNA Ligase/genetics/*metabolism
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Lung Neoplasms/*enzymology/pathology
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*RNA, Small Interfering
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Reverse Transcriptase Polymerase Chain Reaction
8.Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway
Wei ZHOU ; Juan Hua QUAN ; Fei Fei GAO ; Hassan Ahmed Hassan Ahmed ISMAIL ; Young Ha LEE ; Guang Ho CHA
The Korean Journal of Parasitology 2018;56(2):135-145
Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.
Blood Vessels
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Caseins
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Epithelial Cells
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Gene Expression
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Glutamate-Cysteine Ligase
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Glutathione Transferase
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Hydrogen-Ion Concentration
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Phosphoprotein Phosphatases
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Polymerase Chain Reaction
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Reactive Oxygen Species
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Retinaldehyde
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Reverse Transcription
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RNA, Messenger
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Signal Transduction
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Toxoplasma
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Toxoplasmosis
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Vascular Endothelial Growth Factor A
9.Studies on the molecular diagnosis and prenatal diagnosis of the spinal muscular atrophy carriers by multiplex ligation-dependent probe.
Haiyan ZHU ; Yali HU ; Jie LI ; Ying YANG ; Xing WU
Chinese Journal of Medical Genetics 2010;27(1):38-41
OBJECTIVETo study the application of the multiplex ligation-dependent probe amplification (MLPA) method in genetic and prenatal diagnosis for spinal muscular atrophy (SMA).
METHODSFour patients, 16 parents and 4 fetuses from 8 SMA pedigrees were included. MLPA was performed for molecular analysis, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the mutation detection of the 4 patients.
RESULTSFor all the four patients, the same homozygous deletion of the exons 7 and 8 of the survival motor neuron 1 (SMN1) gene, was detected by PCR-RFLP and MLPA. All fourteen parents from the 8 pedigrees were carriers of the SMN1 gene heterozygous deletion, except the mothers in pedigrees 1 and 4 in whom the mutations were different.
CONCLUSIONMLPA is a simple and efficient quantitative method for copy number analysis of the SMN genes. It can be used for the genetic diagnosis and prenatal diagnosis of the SMA patients and carriers.
Adult ; Amplified Fragment Length Polymorphism Analysis ; Exons ; Female ; Genetic Carrier Screening ; Heterozygote ; Humans ; Ligase Chain Reaction ; methods ; Male ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Pedigree ; Polymorphism, Restriction Fragment Length ; Pregnancy ; Prenatal Diagnosis ; methods ; Sequence Deletion ; Survival of Motor Neuron 1 Protein ; genetics ; Young Adult
10.Effects of deltamethrin on gene expression of some antioxidase, gamma glutamylcysteine synthetase and NFE2 related factor 2 (Nrf2) in brain tissue.
Huang-yuan LI ; Nian SHI ; Zhong-hua DAI ; Yu-fang ZHONG ; Si-ying WU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2006;24(5):273-277
OBJECTIVETo study the effects of deltamethrin (DM) on the mRNA expression of copper-zinc dependent SOD (CuZn-SOD), glutathione reductase (GR) and gamma glutamylcysteine synthetase (gamma-GCS) light subunit (GCSl), as well as on expression of both mRNA and protein of gamma-GCS heavy subunit (GCSh) and NFE2 related factor 2 (Nrf2) in cerebral cortex and hippocampus of rats.
METHODSEighteen Wistar male rats were randomizedly divided into three groups, six for each group. The low dosage and high dosage DM treated groups were administrated intraperitoneally with DM (the daily dosage was 3.125, 12.500 mg/kg BWT respectively) for five consecutive days while the control group was administered intraperitoneally with olive oil. The relative amount of mRNA expression of these genes was measured by the method of reverse transcription polymerase chain reaction (RT-PCR) (n = 6). The protein level was detected by the method of immunohistochemistry and image analysis system (n = 4).
RESULTSThere was no change in mRNA expression level of CuZn-SOD, GR, GCSh and Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administrated with DM. However, the mRNA level of GCSl gene in cerebral cortex of high dosage group as well as in both cerebral cortex and hippocampus of the low dosage group was significantly lower than that in corresponding tissue in the control group, and was decreased to 71.1%, 63.6% and 75.2% of mRNA level of corresponding tissue in the control group (P < 0.01). There was no obvious effect on protein level of both GCSh and Nrf2 in CA1, CA2, CA3 and dentate gyrus (DG) of hippocampus as well as on that in cerebral cortex in rats treated with DM.
CONCLUSIONUnder the experimental conditions, there is no obvious effect in the mRNA expression level of CuZn-SOD, GR gene, as well as on expression of both mRNA and protein of Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administered with DM. DM depresses the mRNA expression of GCSl gene, but does not affect the mRNA expression of GCSh gene.
Animals ; Cerebral Cortex ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Glutamate-Cysteine Ligase ; biosynthesis ; genetics ; Glutathione Reductase ; biosynthesis ; genetics ; Hippocampus ; drug effects ; metabolism ; Male ; NF-E2-Related Factor 2 ; biosynthesis ; genetics ; Nitriles ; toxicity ; Pyrethrins ; toxicity ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Superoxide Dismutase ; biosynthesis ; genetics