Gilson Inc의 'Automation of RTqPCR Detection of SARS-CoV-2'를 이용한 응용자료는 한국분석기기(주)에서 제공하였으며 주요 내용은 다음과 같다.
CORONAVIRUS AND COVID-19
“Coronaviruses are enveloped, positive-sense single stranded RNA viruses belonging to the family Coronaviridae and the order Nidovirales and are broadly distributed in humans and other mammals (Huang, et al.,2020).” They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genomesize of coronaviruses ranges from approximately 26 to 32 kilobases, the largest for an RNA virus.
In humans, the viruses cause respiratory infections, including the common cold, which are typically mild, though rarer forms such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) can be lethal. In December 2019, a novel pneumonia outbreak was reported in Wuhan, China (Read, Bridgen, Cummings, Ho, & Jewell,2020) and was later identified to be caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Figure 1). This disease was officially named COVID-19 by the World Health Organization (WHO). Common symptoms of the infection include: respiratory symptoms, fever, cough, shortness of breath, and breathing difficulties. In more severe cases, the infection can cause pneumonia, severe acute respiratory syndrome, kidney failure, and even death.
Figure 1. Rending of a SARS-CoV-2 Viron.
RT-qPCR FOR SARS-COV-2 TESTING
Reverse transcription qPCR (RT-qPCR) combines reverse transcription and qPCR, which is routinely used to detect causative virions from respiratory secretions.
Respiratory samples from humans are obtained and tested as described in the following steps (Figure 2).
Figure 2. RT-qPCR Workflow of COVID-19
PIPETMAX® and RT-qPCR Automation
Gilson’s PIPETMAX automated liquid handler removes the risk of manual error in sample transfers. The liquid handler has a volume range from 1 μL to 1200μL and the ability to use 1 to 8 pipetting tips at any one time (Figure 3). The removable tray can also fit both 96- and 384-well plates. Dual pipetting heads automatically pick the required volume range when running, without changing the heads. With more than one removable tray, researchers can prepare the next experiment while the system is running (Figure 4).
Figure 3. PIPETMAX® with Trilution® micro
Figure 4. PIPETMAX® Removable Tray
RT-qPCR Workflow*
Nucleic Acid Extraction
Performance of RT-qPCR amplification-based assays depend on the amount and quality of sample uploadlate RNA. Viral RNA extraction procedures should be qualified and validated for recovery, purity, and specificity before testing specimens.
Commercially available extraction procedures have been shown to generate highly purified viral RNA when following manufacturer’s recommended procedures for sample extraction.
Quality Control
Due to the sensitivity of RT-qPCR, these assays should be conducted using strict quality control and quality assurance procedures. It’s best practice to use aerosol barrier (Filter) pipette tips only and assay controls should be run concurrently with all test samples.
* The RT-qPCR protocol refers to guidance from the US CDC onFebruary 4th, 2020 and may differ in practice as the situationchanges. This article describes the use of PIPETMAXfor setup ofRT-qPCR plates prior to thermocycling.
PIPETMAX Benefits
PIPETMAX is an ideal solution for the efficient processing of assays. Its small footprint easily fits into any lab. With an appropriate external UV/HEPA hood, it is the ideal tool for qPCR reaction preparation. Additionally, PIPETMAX helps researchers improve the accuracy, reproducibility, and consistency among the viral nucleic acid being processed. PIPETMAX also:
• Helps eliminate user-to-user variability
• Protects researchers from pathogens
• Is built using PIPETMAN® technology for transferring liquids with high accuracy and precision
• Is compatible with PIPETMAN® DIAMOND FilterTips (Figure 5) to offer protection against aerosol contamination
• Offers an open system that’s compatible with many reagents, kits, or protocols
Figure 5. Gilson BLISTER Filter Tips
OTHER AUTOMATED APPLICATIONS
qPCR methods, particularly those utilizing probes and equivalent fluorescent probe hybridization, have been widely adopted in the detection of various pathogens.
Automation of viral nucleic acid extraction and detection has been reported for enteroviruses, hepatitis B, hepatitis C, Epstein-Barr virus, herpes simplex virus, human papillomavirus, influenza B and SARS virus.³
SUMMARY
Gilson liquid handling products, such as PIPETMAX, can be implemented into the RT-qPCR workflow for detection of SARS-CoV-2.
Growing testing demands, expanding assay menus, and a need for increased productivity while ensuring cost-savings and quality resulted in the development of more automated, integrated systems with minimal user interactions in molecular biology laboratories. Gilson automated liquid handling products are implemented in various aspects of the sample workflow, including pre-analytical sample handling, nucleic acid extraction, and PCR plate setup with varying degrees of automation and flexibility, and to improve process efficiency and standardization.
PIPETMAX Specifications
PIPETMAX BUNDLE
Gilson Inc의 'Automation of RTqPCR Detection of SARS-CoV-2'에 대한 궁금한 내용은 본 원고자료를 제공한 한국분석기기(주)를 통하여 확인할 수 있다.
REFERENCES(참고문헌):
1. Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y.,… Cao, B. (2020). Clinical features of patients infected with 2019 novel coronavirus in Wuhan,China. The Lancet, 395 (10223), 497-506. doi:10.1016/s0140-6736 (20)30183-52.
2. Read, J. M., Bridgen, J. R., Cummings, D. A., Ho,A., & Jewell, C. P. (2020). Novel coronavirus 2019-nCoV: early estimation of epidemiological parameters and epidemic predictions. doi: 10.1101/2020.01.23.200185493.
3. Tewari, D., Zellers, C., Acland, H., & Pedersen, J. C. (2007). Automated extraction of avian influenzavirus for rapid detection using real-time RT-PCR.Journal of Clinical Virology, 40(2), 142-145. doi: 10.1016/j.jcv.2007.07.011
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Notice
This application note has been produced and edited using information that was available at the time of publication. This application note is subject to revision without prior notice.
PIPETMAX is a general liquid handing device to be used by trained scientific personnel in general laboratories.
Gilson Application note 1030.
Model Name(모델명): PIPETMAX
The Person in Charge(담당자): An Hyesook
Maker(제조사): Gilson Inc.
Country of Origin(원산지): U.S.A
e-mail: kaisco1@kaisco.co.krData Services(자료제공): Gilson Inc.
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