1.Development of Indicators to Assess the Stability of Remnant Blood Samples Stored in a Biobank: Experience at One Institution.
Sae Hwan KIM ; Young Eun KANG ; Young Jun HONG ; Yoon Hwan CHANG ; Seok Il HONG ; Ae Chin OH ; Jin Kyung LEE
The Korean Journal of Laboratory Medicine 2010;30(6):718-725
BACKGROUND: One of the major concerns with biobanking is the absence of standard operating procedures to eliminate pre-analytical variation arising from sample collection, preparation, and storage. Currently, there is a lack of tools to carry out quality control procedures for stored blood samples. The aim of this study is to assess the quality of stored blood samples in our biobank and to suggest appropriate indicators for their quality control. METHODS: The stored blood samples that we tested have been registered into our biobank since 2003. These were transferred to our biobank after carrying out routine requested tests, because the samples would have otherwise been discarded. For the purpose of quality control, we analyzed the concentrations and the integrity of DNA and RNA extracted from the stored samples and tested the levels of several serum proteins; the results were compared with the corresponding pre-storage levels. RESULTS: A total of 19 samples were stored from 2006 to 2009. Of the 22 samples stored between 2003 and 2005, 50% showed complete DNA integrity. However, sufficient RNA integrity was noted in only 1 sample stored as recently as 2009. High blood urea nitrogen levels were also noted in the stored sera, but the increase did not correlate to the duration of storage. CONCLUSIONS: The amount and integrity of nucleic acids extracted from stored blood samples are potential indicators that can be used for quality control. A guideline for the quality assessment of stored blood samples in a biobank is urgently needed.
Blood Banks/*standards
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Blood Proteins/chemistry/standards
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Blood Urea Nitrogen
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DNA/*analysis/chemistry/standards
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Laboratory Techniques and Procedures
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Quality Control
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RNA/*analysis/chemistry/standards
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Specimen Handling/methods
2.Comparison of the reverse transcription-PCR with the branched DNA assay for measurement of human immunodeficiency virus type 1 RNA levels in plasma of Korean patients.
Dong Il WON ; Jung Yong PARK ; June Myung KIM ; Hyon Suk KIM
Yonsei Medical Journal 2001;42(2):204-208
Viral load testing of human immunodeficiency virus (HIV) is an essential tool for initiating and monitoring the antiretroviral therapy for HIV patients. To this end, several methods including polymerase chain reaction (PCR), branched DNA (bDNA), nucleic acid sequence based amplification assay (NASBA) and internally controlled virion PCR (ICV PCR) have become available. Of these methods, the standard reverse transcription-PCR (RT-PCR) assay has been widely used in Korea. However, no comparison study has been performed among the various detection methods currently used in Korean patients. We evaluated the correlation and agreement between the PCR and the branched DNA (bDNA) assay for measurement of HIV RNA in Korean patients. Eighty randomly selected samples from HIV-1-seropositive patients visiting Yonsei Medical Center Severance Hospital were studied. We found that these assays show good agreement, have a reliable correlation (r = 0.92, mean difference in log10 copies/ mL +/- 2 standard deviation = 0.098 +/- 0.805) and produce values whose relationship is given by the following equation: log10v3 bDNA = -0.3405 + 1.0601 x log10RT-PCR. Thus, we conclude that these two methods may allow direct comparison of the results obtained from different assay systems.
Comparative Study
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DNA/chemistry*
;
DNA/analysis*
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Genetic Techniques/standards*
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HIV-1/genetics*
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Human
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Korea
;
RNA, Viral/blood*
;
Reverse Transcriptase Polymerase Chain Reaction/standards*
3.Study on correlation between ITS sequence of Arctium lappa and quality of Fructus Arctii.
Liang XU ; Deqiang DOU ; Bing WANG ; Yanyun YANG ; Tingguo KANG
China Journal of Chinese Materia Medica 2011;36(14):1931-1935
OBJECTIVETo study the correlation between ITS sequence of Arctium lappa and Fructus Arctii quality of different origin.
METHODThe samples of Fructu arctii materials were collected from 26 different producing areas. Their ITS sequence were determined after polymerase chain reaction (PCR) and quality were evaluated through the determination of arctiin content by HPLC. Genetic diversity, genotype and correlation were analyzed by ClustalX (1.81), Mage 4.0, SPSS 13.0 statistical software.
RESULTITS sequence of A. was obtained from 26 samples, and was registered in the GenBank. Corresponding arctiin content of Fructus arctii and 1000-grain weight were determined. A. lappa genotype correlated with Fructus arctii quality by statistical analysis.
CONCLUSIONThe research provided a foundation for revealing the molecular mechanism of Fructus arctii geoherbs.
Arctium ; chemistry ; genetics ; Computational Biology ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; standards ; Furans ; analysis ; Genotype ; Glucosides ; analysis ; Polymorphism, Genetic ; genetics ; Quality Control ; Sequence Analysis, DNA ; Software
4.DNA identification of Zijingpi's adulterant species Schisandra sphenanthera based on NCBI nucleotide database analysis.
Xiao-li CHENG ; Cai-li LIAO ; Chun-sheng LIU ; Zhen-fang BAI ; Yao-jun YANG ; Jian ZHENG ; Ji ZHANG
China Journal of Chinese Materia Medica 2012;37(17):2534-2537
OBJECTIVETo provide basis for quality control of Zijingpi, DNA identification was used based on NCBI nucleotide database analysis.
METHODFirstly, total DNA of Zijingpi was extracted. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and PCR products was directly sequenced after purification. Finally, ITS sequence similarity and phylogenetic tree were used for identification.
RESULTThe ITS sequence information of the mainstream commercial drugs of Zijingpi was obtained.
CONCLUSIONIt is firstly reported that the mainstream commercial drugs of Zijingpi was the bark of Schisandra sphenanthera.
DNA, Plant ; genetics ; Databases, Nucleic Acid ; Drugs, Chinese Herbal ; chemistry ; standards ; Molecular Sequence Data ; Phylogeny ; Quality Control ; Schisandra ; classification ; genetics ; Sequence Analysis, DNA
5.A new herbs traceability method based on DNA barcoding-origin-morphology analysis--an example from an adulterant of 'Heiguogouqi'.
Xuan GU ; Xiao-qin ZHANG ; Xiao-na SONG ; Yi-mei ZANG ; Li YAN-PENG ; Chang-hua MA ; Bai-xiao ZHAO ; Chun-sheng LIU
China Journal of Chinese Materia Medica 2014;39(24):4759-4762
The fruit of Lycium ruthenicum is a common folk medicine in China. Now it is popular for its antioxidative effect and other medical functions. The adulterants of the herb confuse consumers. In order to identify a new adulterant of L. ruthenicum, a research was performed based on NCBI Nucleotide Database ITS Sequence, combined analysis of the origin and morphology of the adulterant to traceable varieties. Total genomic DNA was isolated from the materials, and nuclear DNA ITS sequences were amplified and sequenced; DNA fragments were collated and matched by using ContingExpress. Similarity identification of BLAST analysis was performed. Besides, the distribution of plant origin and morphology were considered to further identification and verification. Families and genera were identified by molecular identification method. The adulterant was identified as plant belonging to Berberis. Origin analysis narrowed the range of sample identification. Seven different kinds of plants in Berberis were potential sources of the sample. Adulterants variety was traced by morphological analysis. The united molecular identification-origin-morphology research proves to be a preceding way to medical herbs traceability with time-saving and economic advantages and the results showed the new adulterant of L. ruthenicum was B. kaschgarica. The main differences between B. kaschgarica and L. ruthenicum are as follows: in terms of the traits, the surface of B. kaschgarica is smooth and crispy, and that of L. ruthenicum is shrinkage, solid and hard. In microscopic characteristics, epicarp cells of B. aschgarica thickening like a string of beads, stone cells as the rectangle, and the stone cell walls of L. ruthenicum is wavy, obvious grain layer. In molecular sequences, the length of ITS sequence of B. kaschgarica is 606 bp, L. ruthenicum is 654 bp, the similarity of the two sequences is 53.32%.
Berberis
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classification
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cytology
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genetics
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China
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DNA Barcoding, Taxonomic
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methods
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DNA, Plant
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chemistry
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genetics
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DNA, Ribosomal Spacer
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chemistry
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genetics
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Drug Contamination
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Drugs, Chinese Herbal
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isolation & purification
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standards
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Lycium
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classification
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cytology
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genetics
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Medicine, Chinese Traditional
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Phylogeny
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Sequence Analysis, DNA
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Species Specificity
6.Establishment of miniSTR fluorescent detection system and its forensic application.
Yan LIU ; Li LI ; Zhen-Min ZHAO
Journal of Forensic Medicine 2014;30(5):332-336
OBJECTIVE:
To establish miniSTR fluorescent detection system with all detected fragments below 150 bp and to enhance the efficiency of detecting the degraded DNA samples.
METHODS:
All candidate primers were designed by Primer Premier 5 and screened by FastPCR 6.0. The miniSTR multiplex system was established by these selected loci labeling by four fluorescent dye. The parameters of PCR and primer concentrations were subsequently optimized. The electrophoresis was fulfilled under POP4 on 3100-Avant and the typing data was validated by standard DNA 9947A and 007. Fresh blood samples and difficult degraded DNA samples were tested to evaluate the usefulness of the system.
RESULTS:
All amplicons in the established miniSTR fluorescent detection system (D12ATA63, D2S1776, D1GATA113, D4S2408, D17S974, D20S482, D3S3053, Amelogenin, D6S474, D9S1122) were less than 150bp. The profile showed a balanced peak height without extra stutter by optimal protocol. Allele frequencies showed no deviations from Hardy-Weinberg equilibrium. The system showed accumulated probability of discrimination 0.999 999 983 and accumulated triplet excluding probability of paternity 0.996 8. It could detect corrupt muscle tissue, low copy number DNA samples and human tissues fixed by 40% formaldehyde solution for 12 days.
CONCLUSION
The miniSTR fluorescent detection system could be solely used for personal identification of degraded DNA samples or complementally used for paternity tests. And the system could enhance the ability of detecting the trace and degraded DNA.
DNA/chemistry*
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DNA Fingerprinting
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DNA Primers/genetics*
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Electrophoresis, Agar Gel
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Forensic Genetics
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Gene Frequency/genetics*
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Genetic Markers/genetics*
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Genetics, Population
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Humans
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Polymerase Chain Reaction/methods*
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Reference Standards
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Sequence Analysis, DNA/methods*
7.Standardized Sweat Chloride Analysis for the Diagnosis of Cystic Fibrosis in Korea.
Sue Jung KIM ; Mingoo LEE ; Seung Ick CHA ; Hwa Young PARK ; Kang Mo AHN ; Chang Seok KI ; Jeong Ho KIM
The Korean Journal of Laboratory Medicine 2008;28(4):274-281
BACKGROUND: Cystic fibrosis is a chronic progressive autosomal recessive disorder caused by the CFTR gene mutations. It is quite common in Caucasians, but very rare in Asians. Sweat chloride test is known to be a screening test for the cystic fibrosis due to the fact that electrolyte levels in sweat are elevated in patients. In this study, sweat chloride levels in Korean population were measured and analyzed by using standardized pilocarpine iontophoresis sweat chloride test. METHODS: The sweat chloride test was performed in 47 patients referred to Yondong Severance Hospital from August, 2001 to April, 2007 and 41 healthy volunteers. The sweat chloride tests were conducted according to the CLSI C34-A2 guideline using pilocarpine iontophoresis method, and the chloride concentrations in sweat were measured by mercurimetric titration. RESULTS: Four patients showed sweat chloride concentrations higher than 60 mmol/L. Reference interval was calculated as 1.4-44.5 mmol/L by analysis of the results of healthy volunteers (n=41). Four patients who exhibited high sweat chloride levels, had characteristic clinical features of cystic fibrosis and their diagnoses were confirmed either by repeated sweat chloride test or genetic analysis. CONCLUSIONS: Standardized sweat chloride test can be utilized as a useful diagnostic tool for cystic fibrosis in Koreans. In cases of sweat chloride levels higher than 40 mmol/L, the test should be repeated for the possible diagnosis of cystic fibrosis. All the confirmed Korean cases of cystic fibrosis showed sweat chloride level above 60 mmol/L.
Adolescent
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Adult
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Child
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Child, Preschool
;
Chlorides/*analysis/*standards
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Cystic Fibrosis/*diagnosis/genetics
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Female
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Humans
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Infant
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Iontophoresis/methods
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Korea
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Male
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Middle Aged
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Pilocarpine/chemistry
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Sequence Analysis, DNA
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Sweat/chemistry/*secretion
8.Establishment and characterization of an infectious cDNA clone of a classical swine fever virus LOM strain.
Gil Soon PARK ; Seong In LIM ; Seung Ho HONG ; Jae Young SONG
Journal of Veterinary Science 2012;13(1):81-91
Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.
Animals
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Antibodies, Viral/blood
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Base Sequence
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Cell Line
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Classical Swine Fever/immunology/*virology
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Classical swine fever virus/*genetics/immunology/pathogenicity
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Cloning, Molecular
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DNA, Complementary/genetics/immunology
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Immunization/methods/standards/veterinary
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Molecular Sequence Data
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Neutralization Tests/veterinary
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RNA, Viral/chemistry/genetics
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Recombinant Proteins/immunology
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Reverse Transcriptase Polymerase Chain Reaction/veterinary
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Sequence Analysis, DNA
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Specific Pathogen-Free Organisms
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Swine
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Virulence