Earticle

Many DNA samples are exposed to various environments at crime scenes and mixed with other substances that adversely affect STR profiling. In this study, DNA quantification and STR profiling were performed up to 40 weeks with body wash or hand sanitizer mixed with saliva, blood, and semen at a final concentration of 1%. In addition, serological preliminary tests such as SALIgAE, LMG, and AP and immunological confirmatory tests using RSID kits were conducted to examine the effect of body wash and hand sanitizer on three body fluids. Body wash containing surfactants showed more serious effect on DNA quantity and STR quality than hand sanitizer containing ethanol. Saliva was immediately decomposed after mixing hygiene products, making it impossible to analyze STR, however blood and semen produced full STR profiles after 40 weeks of mixing. As a result, saliva was very vulnerable to hygiene products, but blood and semen were not affected until 40 weeks after mixing. In addition, the discrepancy between serological test and STR profiling in blood and semen samples showed that STR analysis should be performed even if the serological test was negative.

법과학 실험실에서는 감정에 적용한 시험방법의 신뢰성을 입증하기 위해서 새로운 시험방법을 도입하고자 하는 경우 반드시 시험방법의 신뢰성을 검증하기 위한 시험방법 유효화가 선행되어야 한다.본 연구에서는 미세한 인체시료가 묻은 흔적 증거물이나 오래된 유해 시료와 같은 소량의 DNA가 존재하는 시료를 위한 정확한 유전자 정량분석법을 도입하기 위하여 Applied Biosystems 사의 Quantifiler DUODNA Quantification kit을 이용하여 실시간 중합효소연쇄반응 기반의 정량분석법에 대한 시험방법 유효화 연구를 진행하였다. Manual protocol과 자동화 장비(Tecan HID EVOlution qPCR/STR setup)를 이용한automated protocol에 대하여 시험방법 유효화 지침에 따라 시험을 진행하였다. 정밀성, 정확성 및 민감성시험결과 Quantifiler DUO DNA Quantification kit은 안정적인 CT 값과 매우 작은 표준편차를 보여 주었다. 이러한 유효화 시험의 결과는 Quantifiler DUO DNA Quantification kit을 이용한 정량방법이 높은 민감도를 가진 정확하고 재현성 있는 시험방법임을 증명하는 것이다.

In the forensic DNA testing laboratories, prior to introducing an existing DNA analysis procedure, reliability of the procedure has to be demonstrated by carrying out internal validation. In order to introduce a sensitive and accurate DNA quantification method for low copy number DNA samples such as trace evidences and old skeletal remains, a validation study of the real time PCR system using the Quantifiler DUO DNA Quantification Kit (Applied Biosystemes) was performed. We have validated protocols for manual and automated Quantifiler DUO setup using the Tecan HID EVOlution qPCR/STR setup. The protocols were validated for Quantifiler DUO DNA quantification according to validation guidelines. Quantifiler DUO DNA Quantification kit showed stable CT values and significantly small standard deviations for precision, accuracy and sensitivity study. The internal validation study has demonstrated that Quantifiler DUO assay is a reliable system with high sensitivity, which gives accurate and reproducible results.

법유전학에서 DNA 정량 과정은 정확한 DNA형을 얻기 위한 필수과정이다. 기존 실시간 증폭 시스템을 이용한 정량 분석법은 시약의 높은 단가와 오랜 시간소요 등과 같은 단점을 가지고 있다. 본 연구에서는 샘플 내 존재하는 특정 인간 DNA양을 측정할 수 있는 신속 DNA 정량 시험법을 개발하 여 기존 시스템의 단점을 보완하였다. 개발된 시험법은 실시간 중합효소 연쇄반응(Realtime PCR) 기술 을 기반으로 Microfluidic chip (Lab-on-a-chip, LOC)을 이용하여 정확한 분석 값을 기존 대비 5배 단축 된 시간(15분) 안에 얻을 수 있었다. 또한, 표준 DNA를 사용하여 시험방법의 정확성, 재현성, 민감성 및 종 특이성을 검사하였고 현장 증거물에 대한 적용 가능성 및 상용 키트에 포함된 정량 시약과의 호 환성을 테스트하여 적용 범위의 다양성을 확인하였다.

Human DNA quantification on DNA profiling is an important process to get clear genotypes without any allele drop-out and peak imbalances in forensic genetics. Conventional realtime PCR for human DNA quantification is a time-consuming and expensive technique. In order to overcome these disadvantages we developed UltraFast Realtime PCR system based on the Microfluidic chip(Lab-on-a-chip, LOC) technology. This system can quickly and precisely measure DNA quantification within 15 minutes which was reducing 5 times than conventional method. We validated the new system based on standard DNA using various measures, such as accuracy, reproducibility, sensitivity, and human specificity. We confirmed it's applicability to the cases of crime scene and it's compatibility with other quantification kits.

There has been increasing interest in standardized and quantitative Epstein-Barr virus (EBV) DNA testing for the management of EBV disease. We evaluated the performance of the Real-Q EBV Quantification Kit (BioSewoom, Korea) in whole blood (WB). Nucleic acid extraction and real-time PCR were performed by using the MagNA Pure 96 (Roche Diagnostics, Germany) and 7500 Fast real-time PCR system (Applied Biosystems, USA), respectively. Assay sensitivity, linearity, and conversion factor were determined by using the World Health Organization international standard diluted in EBV-negative WB. We used 81 WB clinical specimens to compare performance of the Real-Q EBV Quantification Kit and artus EBV RG PCR Kit (Qiagen, Germany). The limit of detection (LOD) and limit of quantification (LOQ) for the Real-Q kit were 453 and 750 IU/mL, respectively. The conversion factor from EBV genomic copies to IU was 0.62. The linear range of the assay was from 750 to 106 IU/mL. Viral load values measured with the Real-Q assay were on average 0.54 log10 copies/mL higher than those measured with the artus assay. The Real-Q assay offered good analytical performance for EBV DNA quantification in WB.

Nuclear DNA contents of spermatia from eight ceramiacean and four dasyacean algae (Ceramiales, Rhodophyta) and microspores from two land plants were estimated by fluorescence microscopic image processing and their nuclear SSU rDNA sequence data were analyzed. In frequency distribution patterns, the DAPI-stained nuclear volume (NV) of spermatia showed two peaks corresponding to 1C and 2C. Nuclear 2C DNA contents estimated from NV were 0.45-2.31 pg in ceramiacean and 0.40-0.57 pg in dasyacean algae and 8.42-9.51 pg in two land plants, Capsicum annuum and Nicotiana tabacum. By nuclear patterning of vegetative cells derived from an apical cell, 2C DNA contents of spermatia were 2.31 pg in an alga having uninucleate and non-polyploid nucleus (Aglaothamnion callophyllidicola), 0.45-1.94 pg in algae having uninucleate and polyploid nucleus (Antithamnion spp. and Pterothamnion yezoense), and 0.40-0.62 pg in algae having multinucleate and non-polyploid nuclei (Griffithsia japonica and dasyacean algae). Each mature spermatium and microspore (pollen grain) seemed to have a 2C nucleus, which may provide a genetic buffering system to protect the genetic content of a spermatium and microspore from potentially lethal mutations. Nuclear DNA content and SSU rDNA sequence of Antithamnion sparsum from Korea were reasonably different from those of Antithamnion densum from France. The data did not support the previous taxonomic studies that these two taxa could be conspecific.

Since its introduction in the forensic field, quantitative PCR (qPCR) has played an essential role in DNA analysis. Quality of DNA should be evaluated before short tandem repeat (STR) profiling to obtain reliable results and reduce unnecessary costs. To this end, various human DNA quantification kits have been developed. Among these kits, the PowerQunat® System was designed not only to determine the total amount of human DNA and human male DNA from a forensic evidence item, but also to offer data about degradation of DNA samples. However, a crucial limitation of the PowerQunat® System is its high cost. Therefore, to minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/2-volume) using DNA samples of varying types and concentrations. Our results demonstrated that the low-volume method has almost comparable performance to the manufacturer's method for human DNA quantification, human male DNA quantification, and DNA degradation index. Furthermore, using a reduced volume of regents, it is possible to run 2 times more reactions per kit. We expect the proposed low-volume method to cut costs in half for laboratories dealing with large numbers of DNA samples.

DNA is known as the genetic material in cells. Various environmental factors can cause DNA damages. One of them is sunlight. The life on earth depends on the sunlight, but on the other hand, the UV light in sunlight can cause skin DNA damages. When these damages are not fully repaired before replication, they can lead to mutations of oncogenes and tumour suppressor gene and result in photo carcinogenesis, in the end, skin cancer.(omitted)

Background: We developed an assay to measure DNA-incorporated 6-thioguanine (DNA-TG) and validated its clinical applicability in Korean pediatric patients with acute lymphoblastic leukemia (ALL) in order to improve individualized thiopurine treatment and reduce the life-threatening cytotoxicity. Methods: The DNA-TG assay was developed based on liquid chromatography-tandem mass spectrometry, with isotope-labeled TG-d3 and guanine-d3 as internal standards. This method was applied to 257 samples of pediatric ALL patients. The DNA-TG level was compared with erythrocyte TG nucleotide (RBC-TGN) level in relation to the TPMT and NUDT15 genotypes, which affect thiopurine metabolism, using Spearman’s rank test and repeated measure ANOVA. Results: For DNA-TG quantification, a linearity range of 10.0-5,000.0 fmol TG/μg DNA; bias for accuracy of ?10.4% ?3.5%; coefficient of variation for intra- and inter-day precision of 3.4% and 5.8% at 80 fmol TG/μg DNA and of 4.9% and 5.3% at 800 fmol TG/μg DNA, respectively; and recovery of 85.7%-116.2% were achieved without matrix effects or carry-over. The median DNA-TG level in the 257 samples was 106.0 fmol TG/μg DNA (interquartile range, 75.8-150.9). There was a strong correlation between DNA-TG and RBC-TGN levels (ρ=0.68, P<0.0001). The DNA-TG/RBC-TGN ratio was significantly higher in NUDT15 intermediate metabolizers (*1/*2 and *1/*3) than in patients with wild-type alleles (P<0.0001). Conclusions: This simple and sensitive method for measuring DNA-TG level can improve therapeutic drug monitoring for thiopurine treatment.

Background: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. Methods: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. Results: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R2=0.916, P<0.0001). Conclusions: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

This study was focused on the genotyping and quantification of Porcine circovirus type 2 (PCV2) in thirty PCV2-positive pigs with different clinical symptoms (PCV2-infected without wasting, PCV2-infected with wasting, PCV2-infected with wasting and lymphoid depletion). The quantity of PCV2 DNA in diverse tissues was significantly differed among these groups. (One-way ANOVA test, p<0.001) Interestingly, PCV2-DNA load in tissues of PCV2-infected pigs without wasting and PCV2-infected pigs with wasting and lymphoid depletion were not significantly differed (p = 0.38), while they were all significantly higher when compared with PCV2-infected pigs with wasting-only. PCV2 DNA quantity in tissues was significantly higher in PCV2a and 2b co-infected pigs compared to the PCV2b only-infected pigs (Wilcoxon test, p = 0.039). The PCV2a and 2b co-infected pigs had increased wasting and lymphoid depletion rate but it was not statistically significant. Therefore, this cross-sectional study suggested that PCV2 DNA load in tissues was diverse by clinical and histological findings. Furthermore, co-infection of PCV2a and 2b affected to the PCV2 DNA load in tissues with increased rate of wasting and lymphoid depletion.

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5% to 95% and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1% for both the approaches, allowing to extend the range of quantifications from 1% to 99%. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.

Standardized cytomegalovirus (CMV) DNA quantification is important for managing CMV disease. We evaluated the performance of the Real-Q CMV Quantification Kit (Real-Q assay; BioSewoom, Korea) using whole blood (WB), with nucleic acid extraction using MagNA Pure 96 (Roche Diagnostics, Germany). Real-time PCR was performed on two platforms: the 7500 Fast real-time PCR (7500 Fast; Applied Biosystems, USA) and CFX96 real-time PCR detection (CFX96; Bio-Rad, USA) systems. The WHO international standard, diluted with CMV-negative WB, was used to validate the analytical performance. We used 90 WB clinical samples for comparison with the artus CMV RG PCR kit (artus assay; Qiagen, Germany). Limits of detections (LODs) in 7500 Fast and CFX96 were 367 and 479 IU/mL, respectively. The assay was linear from the LOD to 106 IU/mL (R2 ≥0.9886). The conversion factors from copies to IU in 7500 Fast and CFX96 were 0.95 and 1.06, respectively. Compared with the artus assay, for values <1,000 copies/mL, 100% of the samples had a variation <0.7 log10 copies/mL; >1,000 copies/mL, 73.3% and 80.6% of samples in 7500 Fast and CFX96, respectively, had <0.5 log10 copies/mL. The Real-Q assay is useful for quantifying CMV in WB with the two real-time PCR platforms.

=목적: 본 연구는 자궁내막암으로 진단 받은 환자에 있어서 DNA 배수성 및 합성기 분율과 임상 조직학적 예후 인자들 중 병기, 분화도, 연령 45세 이상, 조직학적 종류, 자궁근층을 50% 이상 침범된 경우, 림프절 전이 유무들과의 상호 연관성을 알아보고자 하였다.연구 방법: 2000년 1월부터 2004년 11월까지 자궁내막암으로 진단받은 66예를 대상으로 하여 이들 중 41예에서 유세포 분석을 통한 DNA 정량검사와 합성기 분율을 측정하였다. DNA 배수성은 이배수성 종양과 비배수성 종양으로 구분하였고, 합성기 분율은 6% 이상과 미만으로 구분하였다. 이러한 검사는 병기결정을 위한 시험적 개복술이나 진단적 소파술 시 생검을 통해 얻은 신선 조직을 유세포 분석법을 이용해 분석하였다. 결과: 41예의 자궁내막암 환자 중 5예가 비배수성 종양이었고, 16예에서 합성기 분율이 6% 이상이었다. 자궁내막암 환자들의 DNA 배수성을 비교하였을 때, 진행된 병기일수록, 분화도가 증가할수록, 자궁 근층의 침투 깊이가 깊을수록, 조직학적 종류에 따라 비배수성 종양이 유의하게 많음을 알 수 있었다. 나이와 림프절 전이 유무와 DNA 배수성과는 유의한 관계가 없었다. 병기가 진행될수록, 핵 분화도가 높을수록 조직학적 종류에 따라 합성기 분율이 유의하게 높게 나타났다. 나이, 구조 분화도, 자궁 근층으로의 침윤의 깊이, 림프절 전이와 합성기 분율과는 서로 유의한 상관관계가 없었다.

배경 : 최근 PCR의 매 주기마다 증폭산물을 측정하는 실시간(real-time) PCR 방법이 도입되어, 증폭산물을 정확하게 분석하게 되었다. 그러나 실시간 중합효소연쇄반응 검사 도입과 검사 수행시 사용하여야 하는 전용 시약과 소모품의 구입에 많은 비용이필요하므로, 실시간 중합효소연쇄반응 검사 장비가 없는 실험실에서의 사용이 제한적이다. 이에 본 연구에서는 기존의 끝점 PCR을사용하여 전기영동과 브롬화에티듐(EtBr) 염색만으로 신뢰할 수있는 유전자 발현 연구를 수행할 수 있는지를 실시간 중합효소연쇄반응 검사법과의 비교분석을 통하여 평가하고자 하였다. 방법 : 실시간 중합효소연쇄반응 검사를 시행하여 얻은 정량값과 실시간 중합효소연쇄반응 검사 후의 끝점 PCR 산물을 아가로오스겔에 전기영동한 후 브롬화에티듐으로 염색하고 자외선 투시하에서 방출되는 형광강도를 측정하여 정량한 결과로 각각 MMP-1 유전자의 상대정량을 시행하여 비교하였다. 결과 : 실시간 중합효소연쇄반응 검사와 끝점 PCR 산물의 아가로오스겔 전기영동에 의한 MMP-1 유전자의 상대정량 값은 상관계수가 r=0.16 ( P<0.01)으로 매우 낮은상관관계를 나타내었다.

배경 : Hepatitis B virus (HBV) DNA 정량검사는 항바이러스제제의 사용, 치료효과 추적, 내성 HBV의 조기 진단 등 HBV감염의 치료를 위해 필요하다. 최근 검사실에 보급되고 있는 real-time quantitative PCR은 기존의 HBV DNA 검사법의 단점들을 보완하면서 보다 간편하고 정확한 HBV DNA 정량 기법이다.본 연구에서는 최근 국내에서 개발된 Real-Q HBV Quantifica-tion kit의 성능을 평가하였다.방법 : Real-Q HBV Quantification kit의 회수율, 검출 한계,분석특이도 및 진단특이도, 정밀도, 직선성 범위, 그리고 DigeneHybrid-Capture II와 상관성과 internal control (IC)의 간섭영향을 분석하였다.결과 : 평균 %회수율은 108.6% 이었으며, 동일 검체에서 IC과의 경쟁 때문에 target 유전자의 증폭이 억제되지 않음을 확인하였다. Hepatitis C virus (HCV), human immunodeficiency virus(HIV), Cytomegalovirus (CMV) 19개의 양성 검체와 교차반응은 없었으며, 본 검사의 재현성은 검사차례내 변이계수 8.7-11.9%,검사차례간 변이계수 10.5-14.7%, 검사일간 변이계수 13.2-21.4%를 보였다. 검출 한계는 56 copies/mL이었으며, 보고범위는 102-1× 1010 copies/mL이었다, HBV 음성 100검체를 이용하여 분석한 진단특이도는 100%이었고, Digene Hybrid-Capture II와 우수한 상관관계를 보였다(R2=0.9827).

Background : Hepatitis B virus (HBV) DNA quantification is important for the management of HBV infection and identification of the development of resistance. The susceptibility to contamination and more variable reproducibility of results with the conventional HBV DNA quantification method have raised the need of a more simple and accurate method for HBV DNA quantification. Real-time quantitative PCR assays recently introduced in the laboratory can meet these needs. In this study, we evaluated the performance of the Real-Q HBV Quantification kit developed in Korea. Methods : We evaluated the recovery of DNA extraction, the interference of internal control, an analytical sensitivity, specificity, and reproducibility, a clinical specificity, and a reportable range of the Real-Q HBV Quantification kit. The quantification result was also compared to that obtained by the Digene Hybrid-Capture II. Results : The mean percent recovery was 108.6% and there was no interference with the internal control on DNA extraction. None of HIV, hepatitis C virus, or cytomegalovirus showed a cross-reactivity with HBV. This assay detected HBV DNA in a linear range from 102 to 1010 copies/mL, with the detection limit of 56 copies/mL. The assay exhibited a low within-run CV (coefficient of variation) (8.711.9%), between-run CV (10.5-14.7%), and between-day CV (13.2-21.4%). No HBV DNA was detected in any of 100 samples without HBV, resulting in a clinical specificity of 100%. The levels of HBV DNA showed a good correlation with those determined with Digene Hybrid-Capture II (R2=0.9827). Conclusions : The Real-Q HBV Quantification kit showed a good analytical sensitivity, specificity, and high reliability with a broad reportable range. This assay should be clinically useful in managing patients with HBV infection. (Korean J Lab Med 2006;26:442-8)

Assay for the detection and quantification of porcine circovirus type 2 (PCV 2) with the real-time PCR were developed. TaqMan probe real-time using a set of primer/probe was developed for detection of PCV 2. In this study we applied real-time PCR assay to 320 samples, collected from pig farms. In 151 of 320 samples, PCV 2 DNA was detected by conventional PCR assay. All samples positive for PCV 2 DNA in conventional PCR assay were also positive in Real-time PCR assay, but 69 of 169 samples that tested negative for PCV 2 DNA in conventional assay were tested positive in TaqMan probe real-time PCR assay. The test of TaqMan probe real-time PCR resulted in detection and quantification limits of 101 copies per sample. TaqMan probe real-time PCR assay increased the number of samples in which PCV 2 was detected by 21%. TaqMan probe real-time PCR assay is very efficient method in contrast to the conventinal PCR, becoming increasingly important method for gene analysis.