Early diagnosis of leptospirosis of pulmonary diffuse hernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific methed for diagnosis, a genomic library of the main pathoge...Early diagnosis of leptospirosis of pulmonary diffuse hernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific methed for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector PUC9. Recombinant plasmids which have hornologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid,designated PCX7, could detect 1.7 kb fragment of strain 017, 9. 0 kb of strain 601 and 30. 0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and hoptonema illini. The recombinant plasmid PCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.展开更多
Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a...Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.展开更多
In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle （AuNP） probes and a gene chip to detect microRNAs （miRNAs）. Specific oligonucleotides were attached to...In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle （AuNP） probes and a gene chip to detect microRNAs （miRNAs）. Specific oligonucleotides were attached to the glass surface as capture probes for the target miRNAs, which were then detected via hybridization to the AuNP probes. The signal was amplified via the re- duction of HAuCI4 by H202. The use of a single AuNP probe detected 10 pmol L-1 of target miRNA. The recovery rate for miR-126 from fetal bovine serum was 81.5%-109.1%. The biosensor detection of miR-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR （qPCR） results. The use of two AuNP probes further improved the de- tection sensitivity such that even 1 fmol L-t of target miR-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye, The platform simultaneously detected lung cancer related miR-126 and miR-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual miRNA detection.展开更多
A nanoparticle-assembled photonic crystal （PC） array was used to detect single nucleotide polymorphism （SNP）. The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized D...A nanoparticle-assembled photonic crystal （PC） array was used to detect single nucleotide polymorphism （SNP）. The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair （bp） difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease （PKD） with a sensitivity of 0.9 fg/mL （50 aM）-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA （gDNA） extracted from the urine and blood of two PKD-wild type and three PKD positive cats. The standard curves for PKD positive （PKD＋） and negative （PKD-） DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection （LOD） was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction （PCR）. The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analvsis is desirable.展开更多
文摘Early diagnosis of leptospirosis of pulmonary diffuse hernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific methed for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector PUC9. Recombinant plasmids which have hornologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid,designated PCX7, could detect 1.7 kb fragment of strain 017, 9. 0 kb of strain 601 and 30. 0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and hoptonema illini. The recombinant plasmid PCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.
文摘Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.
基金supported by the National Basic Research Program of China (2012CB933303)the National Natural Science Foundation of China (61571429, 61571077, 61401442)+2 种基金the Innovation Team of Henan University of Science and Technology (2015XTD003)the Science and Technology Commission of Shanghai Municipality (12441902600, 1402H233900)the Shanghai Clinical Center/Shanghai Xuhui Central Hospital, Chinese Academic of Sciences (BRC2012002)
文摘In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle （AuNP） probes and a gene chip to detect microRNAs （miRNAs）. Specific oligonucleotides were attached to the glass surface as capture probes for the target miRNAs, which were then detected via hybridization to the AuNP probes. The signal was amplified via the re- duction of HAuCI4 by H202. The use of a single AuNP probe detected 10 pmol L-1 of target miRNA. The recovery rate for miR-126 from fetal bovine serum was 81.5%-109.1%. The biosensor detection of miR-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR （qPCR） results. The use of two AuNP probes further improved the de- tection sensitivity such that even 1 fmol L-t of target miR-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye, The platform simultaneously detected lung cancer related miR-126 and miR-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual miRNA detection.
文摘A nanoparticle-assembled photonic crystal （PC） array was used to detect single nucleotide polymorphism （SNP）. The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair （bp） difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease （PKD） with a sensitivity of 0.9 fg/mL （50 aM）-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA （gDNA） extracted from the urine and blood of two PKD-wild type and three PKD positive cats. The standard curves for PKD positive （PKD＋） and negative （PKD-） DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection （LOD） was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction （PCR）. The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analvsis is desirable.
