• Genomics & Informatics
• /
• 제19권3호
• /
• pp.34.1-34.6
• /
• 2021

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

• 한국진공학회:학술대회논문집
• /
• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
• /
• pp.273-273
• /
• 2013

Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.

• Tuberculosis and Respiratory Diseases
• /
• 제71권4호
• /
• pp.249-253
• /
• 2011

Background: Mycobacterial infection is a problem throughout the world along with the increase of immunocompromised patients. For this reason, there have been many methods for faster and more accurate diagnosis. In this study, we evaluated several laboratory methods for mycobacterial infection. Methods: From January to December 2009, 635 specimens were cultured with mycobacteria growth indicator tube (MGIT) and Ogawa media. Polymerase chain reaction (PCR) was performed with the AdvanSure tuberculosis (TB)/non-tuberculosis mycobacterium (NTM) real-time PCR Kit (LG Life Sciences, Seoul, Korea). The 69 samples showing positive culture results were identified with the AdvanSure Mycobacteria Genotyping Chip Kit (LG Life Science, Seoul, Korea). Results: Sixty-nine (10.9%) out of 635 samples showed positive results for mycobacterial culture. Among the 635 samples, 64 were positive in MGIT, but only 42 were positive in Ogawa media. Of the 635 samples, 607 (95.6%) showed the same results between MGIT and Ogawa and the results of 579 (95.4%) were also consistent with the TB/NTM real-time PCR results. However, in the case of NTM, only one (1/24, 4.2%) was positive in PCR. In the Mycobacteria genotyping chip analysis, the most frequently identified NTM species in descending order were M. avium, M. intracellulare, M. chelonae and M. abscessus. Conclusion: Culturing with a combination of MGIT and Ogawa is recommended to increase the recovery rate of mycobacteria. Although PCR missed a reasonable number of NTM, it is faster and usually gives results that concur with those from the culture. The appropriate combination of diagnostic methods with clinical correlation are necessary.

• 센서학회지
• /
• 제20권3호
• /
• pp.187-192
• /
• 2011

Abrasive blaster is similar to sand blaster, and effectively removes hard and brittle materials. Exiting abrasive blaster has applied to rough working such as deburring and rough finishing. As the need for machining of ceramics, semiconductor, electronic devices and LCD are increasing, micro abrasive blaster was developed, and became the inevitable technique to micromachining. This paper describes the performance of the micro blaster in MEMS process of glass and succeed in domestically producing complete micro blaster. Diameter of hole and width of line in this etching is 100 ${\mu}m$ ~ 1000 ${\mu}m$. Experimental results showed good performance in micro channel and hole in glass wafer. Therefore, this micro blaster could be effectively applied to the micro machining of semiconductor, micro PCR chip.

• 대한전기학회:학술대회논문집
• /
• 대한전기학회 2011년도 제42회 하계학술대회
• /
• pp.59-60
• /
• 2011

본 논문에서는 기존의 PCR 장비가 가지고 있는 낮은 경제성, 장비의 대형화, 긴 분석 시간 등과 같은 단점을 해결하기 위하여 ATMEGA128 마이크로 칩을 사용 continuous-flow PCR 칩의 온도를 제어 하였다. Polydimethylsiloxane (PDMS)와 산화 인듐-주석(Indium tin-oxide, ITO) 유리 기판을 사용하여 continuous-flow PCR 칩을 제작하였고 PDMS를 주조 하여 마이크로 채널을 형성하였다. 또한 유리 기판위에 ITO 전극을 패터닝하여 마이크로 히터를 제작하였다. 이 결과 continuous-flow PCR 칩에서 빠르고 정확한 온도 제어를 통한 DNA 중합 효소 연쇄반응 결과를 얻을 수 있었다.

• BMB Reports
• /
• 제37권5호
• /
• pp.546-551
• /
• 2004

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

• 한국진공학회:학술대회논문집
• /
• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
• /
• pp.88-89
• /
• 2013

A variety of influenza A viruses from animal hosts are continuously prevalent throughout the world which cause human epidemics resulting millions of human infections and enormous industrial and economic damages. Thus, early diagnosis of such pathogen is of paramount importance for biomedical examination and public healthcare screening. To approach this issue, here we propose a fully integrated Rotary genetic analysis system, called Rotary Genetic Analyzer, for on-site detection of influenza A viruses with high speed. The Rotary Genetic Analyzer is made up of four parts including a disposable microchip, a servo motor for precise and high rate spinning of the chip, thermal blocks for temperature control, and a miniaturized optical fluorescence detector as shown Fig. 1. A thermal block made from duralumin is integrated with a film heater at the bottom and a resistance temperature detector (RTD) in the middle. For the efficient performance of RT-PCR, three thermal blocks are placed on the Rotary stage and the temperature of each block is corresponded to the thermal cycling, namely $95^{\circ}C$ (denature), $58^{\circ}C$ (annealing), and $72^{\circ}C$ (extension). Rotary RT-PCR was performed to amplify the target gene which was monitored by an optical fluorescent detector above the extension block. A disposable microdevice (10 cm diameter) consists of a solid-phase extraction based sample pretreatment unit, bead chamber, and 4 ${\mu}L$ of the PCR chamber as shown Fig. 2. The microchip is fabricated using a patterned polycarbonate (PC) sheet with 1 mm thickness and a PC film with 130 ${\mu}m$ thickness, which layers are thermally bonded at $138^{\circ}C$ using acetone vapour. Silicatreated microglass beads with 150~212 ${\mu}L$ diameter are introduced into the sample pretreatment chambers and held in place by weir structure for construction of solid-phase extraction system. Fig. 3 shows strobed images of sequential loading of three samples. Three samples were loaded into the reservoir simultaneously (Fig. 3A), then the influenza A H3N2 viral RNA sample was loaded at 5000 RPM for 10 sec (Fig. 3B). Washing buffer was followed at 5000 RPM for 5 min (Fig. 3C), and angular frequency was decreased to 100 RPM for siphon priming of PCR cocktail to the channel as shown in Figure 3D. Finally the PCR cocktail was loaded to the bead chamber at 2000 RPM for 10 sec, and then RPM was increased up to 5000 RPM for 1 min to obtain the as much as PCR cocktail containing the RNA template (Fig. 3E). In this system, the wastes from RNA samples and washing buffer were transported to the waste chamber, which is fully filled to the chamber with precise optimization. Then, the PCR cocktail was able to transport to the PCR chamber. Fig. 3F shows the final image of the sample pretreatment. PCR cocktail containing RNA template is successfully isolated from waste. To detect the influenza A H3N2 virus, the purified RNA with PCR cocktail in the PCR chamber was amplified by using performed the RNA capture on the proposed microdevice. The fluorescence images were described in Figure 4A at the 0, 40 cycles. The fluorescence signal (40 cycle) was drastically increased confirming the influenza A H3N2 virus. The real-time profiles were successfully obtained using the optical fluorescence detector as shown in Figure 4B. The Rotary PCR and off-chip PCR were compared with same amount of influenza A H3N2 virus. The Ct value of Rotary PCR was smaller than the off-chip PCR without contamination. The whole process of the sample pretreatment and RT-PCR could be accomplished in 30 min on the fully integrated Rotary Genetic Analyzer system. We have demonstrated a fully integrated and portable Rotary Genetic Analyzer for detection of the gene expression of influenza A virus, which has 'Sample-in-answer-out' capability including sample pretreatment, rotary amplification, and optical detection. Target gene amplification was real-time monitored using the integrated Rotary Genetic Analyzer system.

• 한국진공학회:학술대회논문집
• /
• 한국진공학회 2009년도 제38회 동계학술대회 초록집
• /
• pp.245-245
• /
• 2010

최근에 반도체 소자 및 마이크로머신, 바이오센서 등에 사용되는 미세 부품에 대한 연구 개발이 활발히 진행되고 있다. 미세 부품을 제작하기 위한 MEMS 공정은 대표적으로 화학용액을 이용한 습식식각, 플라즈마를 이용한 건식식각 등이 주를 이룬다. Micro blaster는 경도가 강하고 화학적 내성을 가지며 용융점이 높아 반도체 MEMS 공정에 어려움이 있는 기판을 다양한 형태로 식각 할 수 있는 기계적인 식각 공정 기술이라 할 수 있다. Micro blaster의 식각 공정은 고속의 날카로운 입자가 공작물을 타격할 때 입자의 아래에는 고압축응력이 발생하게 되고, 이 고압축 응력에 의하여 소성변형과 탄성변형이 발생된다. 이러한 변형이 발전되어 재료의 파괴 초기값보다 크게 되면 크랙이 발생되고, 점점 더 발전하게 되면 재료의 제거가 일어나는 단계로 이루어진다. 본 연구에서는 micro blaster 장비를 반도체 MEMS 공정에 적용하기 위한 식각 특성에 관하여 확인하였다. Micro blaster 장비와 식각에 사용한 파우더는 COMCO INC. 제품을 사용하였다. Micro blaster를 $Al_2O_3$ 파우더의 입자 크기, 분사 압력, 기판의 종류, 노즐과 기판과의 간격, 반복 횟수, 노즐 이동 속도 등의 공정 조건에 따른 식각 특성에 관하여 분석하였다. 특히 실제 반도체 MEMS 공정에 적용 가능한지 여부를 확인하기 위하여 바이오 PCR-chip을 제작하였다. 먼저 glass 기판과 Si wafer 기판에서의 식각률을 비교 분석하였고, 이 식각률을 바탕으로 바이오 PCR-chip에 사용하게 될 미세 홀과 미세 채널, 그리고 미세 챔버를 형성 하였다. 패턴을 형성하기 위하여 TOK Ordyl 사의 DFR(dry film photoresist:BF-410)을 passivation 막으로 사용하였다. Micro blaster에 사용되는 파우더의 직경이 수${\mu}m$ 이상이기 때문에 $10\;{\mu}m$ 이하의 미세 채널과 미세홀을 형성하기 어려웠지만 현재 반도체 MEMS 공정 기술로 제작 연구되어지고 있는 바이오 PCR-chip을 직접 제작하여 micro blaster를 이용한 반도체 MEMS 공정 기술에 적용 가능함을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권9호
• /
• pp.5519-5525
• /
• 2013

Background: Cervical cancer is the second most common cancer in Thai women after breast cancer. Currently, the Papanicolaou (Pap) smear is the recommended procedure for cervical cancer screening in Thailand, but only a relatively small percentage of women follow this screening program. An alternative method to detect HPV genotypes associated with cervical cancer is self-sampling of urine, which is a more widely accepted method. Our study aimed to evaluate the prevalence of HPV in Thai women using urine and cervical swabs and prevalence of HPV in Thai men using urine samples. Materials and Methods: Tumorigenic HPV detection was accomplished by electrochemical DNA chip and PCR/direct sequencing. In addition to HPV prevalence, we report the concordance between different methods and sample types. One-hundred and sixteen women and 100 men were recruited. Histological examination revealed normal cytology in 52 women, atypical squamous cells of undetermined significance (ASCUS) in 9, low-grade squamous intraepithelial lesions (LSIL) in 24, and high-grade squamous intraepithelial lesions (HSIL) in 31. One-hundred men were classified as heterosexuals (n=45) and homosexuals (n=55). Results: The most prevalent HPV genotype in our study was HPV16. The HPV detection rate was generally lower in urine samples compared with cervical samples. Overall, there was good agreement for the detection of carcinogenic HPV from female cervical samples between the DNA chip and PCR/sequencing, with 88.8% total agreement and a kappa value of 0.76. In male urine samples, the level of agreement was higher in heterosexuals compared with homosexuals. Conclusions: Further improvement is required to increase an overall yield of HPV DNA detection in urine samples before clinical application of a urine-based HPV screening program. The electrochemical DNA chip test is a promising technique for carcinogenic HPV detection.

• The Plant Pathology Journal
• /
• 제30권1호
• /
• pp.51-57
• /
• 2014

A large-scale oligonucleotide (LSON) chip was developed for the detection of the plant viruses with known genetic information. The LSON chip contains two sets of 3,978 probes for 538 species of targets including plant viruses, satellite RNAs and viroids. A hundred forty thousand probes, consisting of isolate-, species- and genus-specific probes respectively, are designed from 20,000 of independent nucleotide sequence of plant viruses. Based on the economic importance, the amount of genome information, and the number of strains and/or isolates, one to fifty-one probes for each target virus are selected and spotted on the chip. The standard and field samples for the analysis of the LSON chip have been prepared and tested by RT-PCR. The probe's specific and/or nonspecific reaction patterns by LSON chip allow us to diagnose the unidentified viruses. Thus, the LSON chip in this study could be highly useful for the detection of unexpected plant viruses, the monitoring of emerging viruses and the fluctuation of the population of major viruses in each plant.