GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address th...GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5’ Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5’RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5’ direction.展开更多
Background Early detection of pulmonary tuberculosis (PTB) is a big challenge in smear negative and sputum scarce patients in China.Simultaneous amplification and testing methods for detection of the Mycobactedum tu...Background Early detection of pulmonary tuberculosis (PTB) is a big challenge in smear negative and sputum scarce patients in China.Simultaneous amplification and testing methods for detection of the Mycobactedum tuberculosis (MTB) complex (SAT-TB assay) is a novel molecular technique established in our hospital.This method has a high sensitivity and specificity in the lab.In this study,the clinical diagnostic performance of this method in smear-negative or sputum-scarce PTB suspects was investigated and evaluated.Methods Two hundred smear negative and 80 sputum-scarce patients were recruited in this study.Samples that included sputum or bronchial washing fluid were collected and sent for both bacteria culture and SAT-TB assay.Diagnosis for these patients was based on the comprehensive evaluation of chestX-ray/CT study,histology examination,lab results,and treatment response.Sensitivity,specificity,positive predictive value (PPV) and negative predictive value (NPV) for each diagnostic test were investigated and calculated using confirmed tuberculosis (TB) and non-TB cases.The time required for detection of MTB was also measured for each method.Results Ninety-two patients (33%) were diagnosed as definitive TB,112 patients (40%) were probable PTB,and 76 (27%) were non-TB.The sensitivity,specificity,PPV,and NPV of SAT-TB in smear-negative PTB suspects were 93% (95% CI,84%-98%),98% (95% CI,90%-100%),98% (95% Cl,91%-100%),and 93% (95% CI,83%-98%).In sputum scarce PTB suspects,the sensitivity,specificity,PPV,and NPV of the SAT-TB assay on bronchial washing fiuids were 90% (95%Cl,74%-98%),100% (95% Cl,85%-100%),100% (95% Cl,88%-100%),and 88% (95% CI,69%-97%).The accuracy of the SAT-TB assay is consistent with the bacteria culture assay.The median time required for detecting MTB in the SAT-TB assay was 0.5 day,which was much faster than bacteria culture (28 days).Conclusions The SAT-TB assay is a fast and accurate method for the detection of MTB.It can be widely applied in the clinic and be an asset in early detection and management of PTB suspects,especially in those patients who are smear negative or sputum scarce.展开更多
Here, we report a novel and universal methodology,termed "ntarctic thermolabile uracil-DNA-glycosylase (AUDG)-supplemented nucleic acid amplification techniques (NAAs) using a labeled-based nanoparticle lateral f...Here, we report a novel and universal methodology,termed "ntarctic thermolabile uracil-DNA-glycosylase (AUDG)-supplemented nucleic acid amplification techniques (NAAs) using a labeled-based nanoparticle lateral flow biosensor (LFB)" (AUDG-NAAs-LFB), which merges enzymatic (AUDG) digestion of contaminant amplicons with different nucleic acid amplification techniques (NAAs), and uses a lateral flow biosensor (LFB) for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets (two amplicons generated from amplification of target sequences, and a chromatography control), without the need for probe- hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification (MCDA), which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA- LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof- of-concept method (AUDG-MCDA-LFB) can be easily reconfigured to detect various nudeic acid sequences by redesigning the specific MCDA primers.展开更多
基金foundations from Chinese Academy of Sciences and Special Funds for Major State Basic reseaxch of China (G1999053903).
文摘GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5’ Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5’RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5’ direction.
文摘Background Early detection of pulmonary tuberculosis (PTB) is a big challenge in smear negative and sputum scarce patients in China.Simultaneous amplification and testing methods for detection of the Mycobactedum tuberculosis (MTB) complex (SAT-TB assay) is a novel molecular technique established in our hospital.This method has a high sensitivity and specificity in the lab.In this study,the clinical diagnostic performance of this method in smear-negative or sputum-scarce PTB suspects was investigated and evaluated.Methods Two hundred smear negative and 80 sputum-scarce patients were recruited in this study.Samples that included sputum or bronchial washing fluid were collected and sent for both bacteria culture and SAT-TB assay.Diagnosis for these patients was based on the comprehensive evaluation of chestX-ray/CT study,histology examination,lab results,and treatment response.Sensitivity,specificity,positive predictive value (PPV) and negative predictive value (NPV) for each diagnostic test were investigated and calculated using confirmed tuberculosis (TB) and non-TB cases.The time required for detection of MTB was also measured for each method.Results Ninety-two patients (33%) were diagnosed as definitive TB,112 patients (40%) were probable PTB,and 76 (27%) were non-TB.The sensitivity,specificity,PPV,and NPV of SAT-TB in smear-negative PTB suspects were 93% (95% CI,84%-98%),98% (95% CI,90%-100%),98% (95% Cl,91%-100%),and 93% (95% CI,83%-98%).In sputum scarce PTB suspects,the sensitivity,specificity,PPV,and NPV of the SAT-TB assay on bronchial washing fiuids were 90% (95%Cl,74%-98%),100% (95% Cl,85%-100%),100% (95% Cl,88%-100%),and 88% (95% CI,69%-97%).The accuracy of the SAT-TB assay is consistent with the bacteria culture assay.The median time required for detecting MTB in the SAT-TB assay was 0.5 day,which was much faster than bacteria culture (28 days).Conclusions The SAT-TB assay is a fast and accurate method for the detection of MTB.It can be widely applied in the clinic and be an asset in early detection and management of PTB suspects,especially in those patients who are smear negative or sputum scarce.
文摘Here, we report a novel and universal methodology,termed "ntarctic thermolabile uracil-DNA-glycosylase (AUDG)-supplemented nucleic acid amplification techniques (NAAs) using a labeled-based nanoparticle lateral flow biosensor (LFB)" (AUDG-NAAs-LFB), which merges enzymatic (AUDG) digestion of contaminant amplicons with different nucleic acid amplification techniques (NAAs), and uses a lateral flow biosensor (LFB) for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets (two amplicons generated from amplification of target sequences, and a chromatography control), without the need for probe- hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification (MCDA), which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA- LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof- of-concept method (AUDG-MCDA-LFB) can be easily reconfigured to detect various nudeic acid sequences by redesigning the specific MCDA primers.