MGI Easy Nucleic Acid Extraction Kit Assignment

Assignment Task

Introduction

Biogroup is first owned by a French group and exclusively managed by biologists, the business knowledge and professionalism are the main strength. The innovation, excellence and by the common values of expertise and their proximity by uniting, increased the efficiency of the diagnosis to treat patients faster. The founder of the company is DR. Stephane Eimer started up in the year 1998. In 2020 Covid -19 become a challenge and hard to fight all over the world. In that pandemic situation in 2020 French group stepped into the UK and expanded their clinics and Biogroup pathology laboratory in London. Initially started up with the SARS-COV-2 testing(RT-PCR) method and antibody testing. Around 7000 to 8000 samples have been processed per day. The purpose of start-up describes the process of performing qualitative Real-time PCR analysis in detecting the presence of SARS-CoV2 (COVID-19) viral RNA in patient respiratory samples. It provides information on how to use the BioRad CFX96 Real-time PCR detection system and MGISP-960 automatic nucleic acid extraction instrument in the analysis process.

 The Eurobio Plex EBX-041 diagnostic kit is used for the qualitative detection of SARS-CoV-2 using reverse-transcription and real-time amplification of extracted RNA material and using specific primers for the amplification of specific target protein genes of the viral genome.

The RT-PCR method detects three specific genes: RNA-dependent RNA polymerase target genes RdRp1 gene and RdRp2 gene, and target 3 on the N gene. The presence of the SARS-CoV-2 gene is detected with specific probes of each target, labelled respectively with FAM (RdRp1 gene), HEX (RdRp2 gene) and Texas red (N gene). During the extension phase of RT-PCR, the probes emit specific fluorescence following hydrolysis during elongation of the amplification product. The measurement of the intensity of real-time fluorescence correlates with the accumulation of amplification products.

Equipment used in detecting the SARS-COV-2 at Biogroup laboratory:

• Biosafety cabinets
• Centrifuges
• Hamilton Vantage for sample extraction
• MGI for the whole PCR extraction process
• CFX -thermal cycler to check the real-time amplification
• Vita machines

Covid 19

Coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. Most of the people infected with the virus need medical attention due to the severity and some experienced mild to moderate respiratory illness and recovered with medication, older people and people with cardiac disease, diabetes, and chronic respiratory disease are very much likely to develop the serious illness, initially, the death rate was high and according to UK government strategy, 315409,116 covid -19 cases been recorded in the United Kingdom. The virus is contagious, and the most common symptoms are Fever, Cough, loss of taste and smell, headache difficulty in breathing/ shortness of breath.

Detection of Covid 19

The diagnostic method is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV2 by amplification of specific viral protein genes.

The assay is composed of two principal steps:

1. Extraction of RNA using the MGI Easy Nucleic Acid Extraction Kit and processing on the MGISP-960 sample preparation system.
2. Reverse transcription and PCR amplification was performed on the BIORAD CFX96 Real-Time PCR Detection System.

The Reverse transcription polymerase chain reaction (RT-PCR) is a technique used to identify the presence of specific genetic material through the process of amplification using enzymes and is based on specific target recognition. The initial step in RT-PCR for SARS-CoV-2 involves the extraction of the viral RNA. The purified extract is then added to a reaction mixture that includes primers (short stretches of nucleic acid specific to the target region of the viral genome), nucleotide bases (building blocks of nucleic acids), enzymes (initiates and complete the reaction) and fluorescent labelled probes (nucleic acid that binds to reaction product and acts as reaction indicator).

The primers and probes are contained in the oligomix for the detection of the 3 target genes. The N primer is specific to COVID-19 and detects a region in the nucleocapsid gene, and the RdRp1 & 2 targets the polymerase gene and is common with the SARS virus. The Oligomix also contains an endogenous human control gene and corresponding Cy5 labelled probe, which serves as an internal control for the test, allowing the evaluation of correct nasopharyngeal sampling.

The primers attach to target regions of the viral nucleic acid, allowing the enzymes to add nucleotides to elongate the complementary DNA strand. The sample reaction mixture is subjected to repeated thermal cycles so that copies of the viral target are doubled per cycle leading to exponential rise and reaction indicated by labelled probes emission of fluorescent signal. The earlier occurrence of exponential increase is proportional to the quantity of virus in the sample. The reaction is determined by the cycle threshold (Ct) value (figure 1) which demonstrates the thermal cycle number at which the fluorescent signal exceeds that of the background and passes the threshold for positivity.

Figure 1. Illustration of Cycle threshold values for qualitative real-time PCR gene amplifications.

(CT values 28.04 Cy5 internal control, 26.97 FAM RdRp1, 28.98 HEX RdRp2, 32.01 Texas Red N gene)

Table 1. Qualitative Real-Time PCR setting for version 11 Eurobioplex SARS-CoV-2 detection on Bio-Rad CFX96 instrument

Activities:

• My role in the Biogruop laboratory is to receive and check samples and request forms reaching the laboratory from our service users
• To label samples (where required) and forms with a unique identifier and place specimen containers in processing racks
• Entering patient details onto the KALISIL laboratory information management system (LIMS), where required
• To liaise with users regarding errors in patient and specimen identification, returning samples to the sender when appropriate, and recording errors
• Maintenance of a high standard of work, checking specimen and request details, any non-conformity brought to the attention of a senior member of staff
• To always comply with the BioGroup Laboratory health and safety procedures and laboratory codes of practice and adhere to an organisation and departmental policies and procedures
• To follow laboratory procedures as detailed in departmental standard operating procedures (SOP)
• Assist in the training of new starters
• To carry out duties within the molecular department as and when required by the Laboratory Manager
• Receipt of COVID-19 RT-PCR samples
• Preparation of PCR series
• Launch of PCR series
• Management of PLCs and their maintenance
• Technical validation of the results
• Participation in the drafting of quality documents and method validations

Identification of SARS -COV-2 using PCR method

Work Flowchart:

Receipt of the sample, plate plan and sampling:

Sample tubes not already registered on kalisil/ LIMS(Laboratory Information Management System) upon arrival are registered by technicians and stored at 5 degrees Celsius in the fridge.

The barcode sample tubes are scanned into CFX software which creates a plate plan in CSV file format for the series, the plate plan covers 96 deep wells 93 being a patient sample and 3 wells for controls. The plate plan is saved and printed as a pdf and attached to the cover sheet. The file is also saved as a  CSV file on the plate plan software.

As per infection control policy the sampling was carried out manually in the laminar airflow of the biosafety cabinet.

180ul of each patient sample is pipetted to the deep 96well plates, care is taken to avoid cross-contamination of each well and at the last 3 wells, the controls are to be added.

Reagent preparation:

Add the listed following components to a beaker,

Close the beaker and invert 10 times to mix and dispense 360ul of buffer reagent into each well except for the positive and negative controls.

Launching of MGI:

The MGI script and the whole process are saved in the software all we need to do is just select the script for 1 series or 2 and need to start the run, the whole nucleic acid extraction with Eurobio RT-qPcr method is saved and the process carried out automatically. The process takes 45minutes to complete.

Preparation of Master mix:

Before preparing the master mix the tubes should be homogenised, Enzyme taq polymerase and enzyme reverse transcriptase must be gently inverted 10 times Oligomix and CI-RNA must be vortexed for 10 seconds at the lowest speed Water and positive control are vortexed for 5 seconds at the lowest speed.

The volume of master mix used for 1 plate is listed below

The above volume is prepared and mixed well,20ul of the master mix is added into each well of the qPCR plate including the wells of 3 controls. Now the 5ul of RNA of each patient sample is transferred to qPCR plates which contain 20ul of master mix per well.

CFX launch:

Centrifuge the RNA containing qPCR plates for 2 minutes and open the CFX software and click on the CFX software in which the series will be launched and locate the CSV for the series by opening the shortcut and drag the file into the CFX software and start run and it takes 50 minutes for the whole process.

Validation:

During validation 3 controls are checked for conformity to the expected Ct values and curves. If the results of the controls do not confirm, a non-conformity is created on Kalisil and it is reported to the biologist so that it can be taken into account for biological validation.

Results Export:

The fluorescence shift baseline settings should be selected, and the file is saved before exporting the results to the patients. Save the export file into COVTOSIL and select the saved file from the COVTOSIL folder and generate the results and the files will automatically be uploaded to Kalisil and sent to the patient.

Achievements:

Throughout the placement year, I have got an opportunity to expose myself to a good work environment. My communication skills, interpersonal skills have improved a lot. At the busiest time worked a continuous shift with full energy and got an appreciation for my hard work and dedication from my management. I have learnt good documentation practice by maintaining them confidentially. During my employment, the organisation provided more training and encourage me to do online certification courses related to HB compliance and I am glad to say that have completed all the training successfully on time. Taken responsibility to provide good training to the new technicians from basic steps. Helped my senior team and biologist by drafting the documents, collecting the data of QC controls and repeatability of the test. Taken responsibility for doing weekly maintenance and cleaning of all equipment at the lab by signing the log sheet. Assisted in Clinical and clerical duties that increased overall efficiency.

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