ABSTRACT
OBJECTIVE: To establish a rapid and accurate high performance liquid chromatography/mass spectrometry method for the analysis of impurities in digoxin crude drug. METHODS: An HPLC-ESI-IT-TOF method was established. The fragmentation pathways of digoxin and digitoxin standards were studied. RESULTS: A total of 12 impurities were identified or tentatively characterized based on the fragmentation pathway. Digoxigenine-bisdigitoxoside-mono-glucose digoxigenin-tridigitoxosido-monoglucose were found for the first time in digoxin. CONCLUSION: It is extremely simple using HPLC-ESI-IT-TOF to provide chemical information concerning the constituents in Chinese herbal medicines, and it makes the identification results more convinced. The method has been applied for the international comparison study and given good results. Copyright 2012 by the Chinese Pharmaceutical Association.
ABSTRACT
Two non-radioactive probes using digoxigenin or biotin were developed for detecting canine herpesvirus (CHV) and compared for their sensitivities by in situ hybridization (ISH) in formalin fixed, paraffin embedded sections, which has been used routinely in veterinary fields. Sections of the CHV-infected cell preparation were subjected to several different ISH protocols using digoxigenin- or biotin-labeled probe respectively. Results were compared for the hybridization and background signal intensities. The best result was obtained by the optimized ISH protocol using digoxigenin-labeled probe for detection of CHV DNA. The optimized ISH assay, which developed in this study, may be a valid tool for the study of pathogenesis and diagnosis of CHV infection.
Subject(s)
Animals , Dogs , Biotin , Cell Line , DNA Probes/chemistry , DNA, Viral/chemistry , Digoxigenin , Dog Diseases/diagnosis , Herpesviridae Infections/diagnosis , Herpesvirus 1, Canid/genetics , In Situ Hybridization/methods , Polymerase Chain Reaction/veterinary , Sensitivity and SpecificityABSTRACT
The ribosomal DNA fragment was amplified with PCR utilizing synthetic primers with host bacteria chromosomal DNA as template. The resultant probes were labeled with Digoxigenin and were applied in the dot blot of DNA vaccine samples. The host bacteria genomic DNA in DNA vaccine against hepatitis B was less than 15 ng/?g. Digoxigenin labeled probes proved sensitive and reliable in determining genomic DNA.
ABSTRACT
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) was found in several serotypes of E coli including 0157 serotype. Sorbitol-MacConkey agar: may be useful for the detection of E. coli 0157, but is not helpful for the detection of sorbitol-fermenting STEC other than 0157. Moreover, some strains of E. colt 0157 can ferment sorbitol. In this study, in situ hybridization using DNA probe of shiga toxin was used for the isolation of STEC from the PCR-positive stool and -Sequenbe analysis of a part of shiga toxin gene was performed. METHODS: The stool was incubated in LB broth overnight and DNA was extracted from the culture fluid. Multiplex PCR was performed with primers for stxl and stx2 genes. Specimen showed PCR-positive was incubated on MacConkey agar and colonies were blotted with nitrocellulose membrane. Digoxigenin-labelled DNA probe for shiga toxin was made by PCR and the positive colonies were detected with anti-digoxigenin-alkaline phosphatase conjugate and nitroblue tetrazolium. Agglutination test with antisera was performed for the serotying and VTEC-RPLA kit was used for the toxin production. Sequence analysis of PCR products was performed with automatic sequence analyser. RESULTS: An stxl-negative, but stx2-positive PCR was observed in a three-year-old girl, who visited Kumi Hospital on July 19, 1999 complaining of vomiting and diarrhea. The positive colonies were isolated by in situ hybridization using stx2-specific DNA probe. The titers of stxl and stx2 by VTEC-RPLA test were negative and 1:64, respectively. Agglutination for the serotyping was not observed with all of the 0 antisera. 160-nucleotide sequence of stx2 of this isolate was identical with bacteriophage 933W (GenBank X07865), except for the change (T-C) of 957th nucleotide and amino acid sequence was identical each other. CONCLUSIONS: For the sensitive detection of STEC from the stool of patients with diarrhea, multiplex PCR is recommended with stxl- and stx2-specific primers. And in situ hybridization should be performed in PCR-positive specimen for the isolation of STEC. This method may be helpful for the detection of STEC as the causative microorganisms in food-borne outbreak.
Subject(s)
Female , Humans , Agar , Agglutination , Agglutination Tests , Amino Acid Sequence , Bacteriophages , Collodion , Diarrhea , DNA , Escherichia coli , Immune Sera , In Situ Hybridization , Membranes , Multiplex Polymerase Chain Reaction , Nitroblue Tetrazolium , Polymerase Chain Reaction , Sequence Analysis , Serotyping , Shiga Toxin , Shiga-Toxigenic Escherichia coli , Sorbitol , VomitingABSTRACT
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) was found in several serotypes of E coli including 0157 serotype. Sorbitol-MacConkey agar: may be useful for the detection of E. coli 0157, but is not helpful for the detection of sorbitol-fermenting STEC other than 0157. Moreover, some strains of E. colt 0157 can ferment sorbitol. In this study, in situ hybridization using DNA probe of shiga toxin was used for the isolation of STEC from the PCR-positive stool and -Sequenbe analysis of a part of shiga toxin gene was performed. METHODS: The stool was incubated in LB broth overnight and DNA was extracted from the culture fluid. Multiplex PCR was performed with primers for stxl and stx2 genes. Specimen showed PCR-positive was incubated on MacConkey agar and colonies were blotted with nitrocellulose membrane. Digoxigenin-labelled DNA probe for shiga toxin was made by PCR and the positive colonies were detected with anti-digoxigenin-alkaline phosphatase conjugate and nitroblue tetrazolium. Agglutination test with antisera was performed for the serotying and VTEC-RPLA kit was used for the toxin production. Sequence analysis of PCR products was performed with automatic sequence analyser. RESULTS: An stxl-negative, but stx2-positive PCR was observed in a three-year-old girl, who visited Kumi Hospital on July 19, 1999 complaining of vomiting and diarrhea. The positive colonies were isolated by in situ hybridization using stx2-specific DNA probe. The titers of stxl and stx2 by VTEC-RPLA test were negative and 1:64, respectively. Agglutination for the serotyping was not observed with all of the 0 antisera. 160-nucleotide sequence of stx2 of this isolate was identical with bacteriophage 933W (GenBank X07865), except for the change (T-C) of 957th nucleotide and amino acid sequence was identical each other. CONCLUSIONS: For the sensitive detection of STEC from the stool of patients with diarrhea, multiplex PCR is recommended with stxl- and stx2-specific primers. And in situ hybridization should be performed in PCR-positive specimen for the isolation of STEC. This method may be helpful for the detection of STEC as the causative microorganisms in food-borne outbreak.
Subject(s)
Female , Humans , Agar , Agglutination , Agglutination Tests , Amino Acid Sequence , Bacteriophages , Collodion , Diarrhea , DNA , Escherichia coli , Immune Sera , In Situ Hybridization , Membranes , Multiplex Polymerase Chain Reaction , Nitroblue Tetrazolium , Polymerase Chain Reaction , Sequence Analysis , Serotyping , Shiga Toxin , Shiga-Toxigenic Escherichia coli , Sorbitol , VomitingABSTRACT
AIM: To detect CD23 mRNA expression in B-cell lymphoma. METHODS: 20 pathological diagnostic conformed B-cell lymphoma samples were selected. Surface CD23 protein expression was examined by SP-immunohistochemistry and CD23 mRNA was detected by in situ hybridization with digotoxin labeled CD23 DNA probe.RESULTS: The positive rates of both surface CD23 protein and CD23 mRNA expression in B-cell lymphoma samples were more than 90%. CONCLUSIONS: The high expression of CD23 was showed in B-cell lymphoma both in mRNA and in protein levels. The results of this study was useful for understanding the molecular mechanism of B-cell lymphoma and for clinical diagnosis and treatment of the disease.
ABSTRACT
The technique of in situ hybridization using synthetic oligonucleotides labelled by non-radioactive method was developed to localize vasoactive intestinal polypeptide, arginine-vasopressin and oxytocin mRNAs in the rat brain. Also double in situ hybridization technique where combination of non-radioactive and radioactive probes were applied was developed to localize 2 neuropeptide mRNAs in single tissue section. The results were as follows; In non-radioactive in situ hybridization methods using digoxigenin-labelled oligonucleotide probe, alkaline-phosphates method using NBT and BCIP as substrates gave the best result that specific hybridization signals were observed. In radioactive in situ hybridization methods using 35S-labelled oligonucleotide probe, specific hybridization signals were observed in both nuclear track emulsion and X-ray film autoradiography. In double in situ hybridization methods using combination of 35S-labelled and digoxigenin-labelled oligonucleotide probes, specific hybridization signals were observed in the group where K5 emulsion was applied as nuclear track emulsion. The technique of in situ hybridization using digoxigenin-labelled oligonucleotide applied in this study will be useful as alternative for radioactive in situ hybridization technique. Moreover, combination of non-radioactive and radioactive labelled probes in double in situ hybridization technique will be a useful tool for the simultaneous localization of various mRNAs in single section for the study of various neurotransmitters, neuropeptides, receptors and signal transduction molecules.
Subject(s)
Animals , Rats , Aging , Autoradiography , Brain , Corpus Callosum , Digoxigenin , In Situ Hybridization , Magnetic Resonance Imaging , Mesencephalon , Neuropeptides , Neurotransmitter Agents , Oligonucleotide Probes , Oligonucleotides , Oxytocin , Pons , RNA, Messenger , Signal Transduction , Vasoactive Intestinal Peptide , Vasopressins , X-Ray FilmABSTRACT
In situ hybridization was performed in ten cases of condyloma acuminata in order to study the applicability of digoxigenin-11 dUTP(Dig-dUTP) labelled probe compared with radioactive isotope labelled probes. Although signal intensity was denser in radiolabelled probes, high positive rates were obtained with Dig-dUTP labelled probes. From these results, Dig-dUTP labelling is found to be more efficient in typing of human papillomavirus DNA than radiolabelling.
Subject(s)
Humans , DNA , In Situ HybridizationABSTRACT
In situ hybridization was performed in ten cases of condyloma acuminata in order to study the applicability of digoxigenin-11 dUTP(Dig-dUTP) labelled probe compared with radioactive isotope labelled probes. Although signal intensity was denser in radiolabelled probes, high positive rates were obtained with Dig-dUTP labelled probes. From these results, Dig-dUTP labelling is found to be more efficient in typing of human papillomavirus DNA than radiolabelling.
Subject(s)
Humans , DNA , In Situ HybridizationABSTRACT
A new method to revel RFLPs is presented. The human genomic DNAwas purified by saturatedNaCl solution and the pAW101 probe labelled with digoxigenin-dUTP. The relationships of RFLPsand genetic patterns of PGM1 (phosphoglucomutase),EsD (esterase D),GLO1 (glyoxalase)and ACP(acid phosphatase ) between the fillal generation and parental generation were detected in 15 families(among them 11 cases were aborted fetuses). The probability of paternity (w)was caculated accor-ding to Essen - Moller's formula, each w vlua was over 99. 73 %, reached the standard of incladingpaternity. An effective,rapid, and non-toxic RFLPs technique was established, which is easy to man-age in common lab oratories.
ABSTRACT
The TNA mRNA expression in 33 samples of cervical carcinoma and 28 samples of condyloma have been observed by using ~3H and Digoxigenin labeled TNF—? cDNA probes and in situ hybridization technique, we found that the results of in situ hybrdfzation with these two kinds of labeled probes were alike, the sensitivity with ~3H labeled probe was slightly higher than that with Digoxigenin labeled probe. Nevertheless, the nonisotope probe would be used more and more in future because of its safety, rapidity and convenience in work.