ThermoFisher TaqPath Assay vs RT-PCR: Detection for Omicron: Types of Coronavirus Tests YouTube Lecture Handouts

Doorsteptutor material for competitive exams is prepared by world's top subject experts: get questions, notes, tests, video lectures and more- for all subjects of your exam.

Types of Coronavirus Tests

ThermoFisher TaqPath Assay vs RT-PCR: Detection for Omicron: Types of Coronavirus Tests|Covid-19

Illustration: Types of Coronavirus Tests

Swab Test – In this case, a special swab is used to take a sample from your nose or throat

Nasal aspirate – In this case, a saline solution will be injected into your nose and, then a sample is taken with a light suction

Tracheal aspirate – In this case, a thin tube with a torch, also known as a bronchoscope, is put into your mouth to reach your lungs from where a sample is collected.

Sputum Test – Sputum is thick mucus that gets accumulated in the lungs and comes out with a cough. During this test, you՚re required to cough up sputum in a special cup or a swab is used to take a sample from your nose.

Blood test – In this case, a blood sample is taken from a vein in the arm.

RT PCR

SGTF (S Gene Target Failure)

The World Health Organization has said that in the RT-PCR tests targeting three genes (N, S and E) , the S gene is not present in the case of Omicron mutations, therefore, the absence of՚s gene dropout ‘or՚s gene target failure’ (SGTF) can be used as the marker for this variant, pending genome sequencing confirmation.

Thermo Fisher TaqPath RT-PCR kits with higher specificity and better detection rate to the labs that have superior access.

TaqPath tests will be used for testing samples of reinfection, vaccine breakthrough infections, unusual transmission events, positive children, severity of illness in patients without comorbidities.

According to the international organization, similar to the alpha variant, the S gene target failure (SGTF) or dropout due to deletion at Spike position 69 - 70 has been reported in the new variant Omicron

Illustration: RT PCR

TaqPath

Thermo Fisher Scientific has worked rapidly to develop a multiplex real-time PCR diagnostic kit to enable clinical and public health laboratories to quickly diagnose COVID-19 caused by SARS-CoV-2 infection.

ORF1ab (also ORF1a⟋b) refers collectively to two open reading frames (ORFs) , ORF1a and ORF1b, that are conserved in the genomes of noroviruses, a group of viruses that includes coronaviruses. The genes express large polyproteins that undergo proteolysis to form several nonstructural proteins with various functions in the viral life cycle, including proteases and the components of the replicase-transcriptase complex (RTC) . The two ORFs are related by a programmed ribosomal frameshift that allows the ribosome to continue translating past the stop codon at the end of ORF1a, in a -1-reading frame. The resulting polyproteins are known as pp1a and pp1ab.

ORF1a is the first open reading frame at the 5′ end of the genome. Together ORF1ab occupies about two thirds of the genome, with the remaining third at the 3′ end encoding the structural proteins and accessory proteins

Table Supporting: TaqPath
AssayTarget gene (position in SARS-CoV-2 genome)Internal controlThermal cycling conditionPCR reagentReaction time, minutes
CDC N1 kitN (28,286 to 28,357)RNaseP in separate reaction10 minutes at , 2 minutes at , 45 cycles of 3 seconds at and 30 seconds at TaqPath88
CDC N2 kitN (29,163 to 29,229)RNaseP in separate reaction10 minutes at , 2 minutes at , 45 cycles of 3 seconds at and 30 seconds at TaqPath88
CDC RP kitHuman RNaseP-10 minutes at , 2 minutes at , 45 cycles of 3 seconds at and 30 seconds at TaqPath88
NIID N2N (29,142 to 29,280)None15 minutes at , 2 minutes at , 45 cycles of 3 seconds at and 30 seconds at TaqPath68
NIID N2 with EAVN (29,142 to 29,280)EAV Extraction Control kit15 minutes at , 2 minutes at , 45 cycles of 3 seconds at and 30 seconds at TaqPath68
Corman EE (26,268 to 26,380)None15 minutes at , 2 minutes at , 45 cycles of 3 seconds at and 30 seconds at TaqPath68
Corman NN (28,555 to 28,682)None15 minutes at , 2 minutes at , 45 cycles of 3 seconds at and 30 seconds at TaqPath68
Roche E kitEEAV Extraction Control kit5 minutes at , 5 minutes at , 45 cycles of 5 seconds at , 15 seconds at , and 15 seconds at Light Cycler65
Roche RdRP kitRdRPEAV Extraction Control kit5 minutes at , 5 minutes at , 45 cycles of 5 seconds at , 15 seconds at , and 15 seconds at Light Cycler65
Roche N kitNEAV Extraction Control kit5 minutes at , 5 minutes at , 45 cycles of 5 seconds at , 15 seconds at , and 15 seconds at Light Cycler65
Thermo Combo kitORF1ab, S, NMS2 phage extraction control10 minutes at , 2 minutes at , 40 cycles of 3 seconds at and 30 seconds at Included67
BGI kitORF1ab (3180 to 3280)Human beta-actin gene20 minutes at , 10 minutes at , 40 cycles of 15 seconds at and 30 seconds at Included90
LAMP kitRdRP, NNot included35 minutes at Included35
  • Real-time RT-PCR by N1, N2, and RNaseP (RP) internal control assays developed by the Centers for Disease Control and Prevention (2019-nCoV CDC EUA kit, obtained from Integrated DNA Technologies, Coralville, IA) , N2 assay developed and distributed by the NIID in Japan (with⟋without EAV) , and N and E assays developed by Charite in Germany (Corman) with TaqPath 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific) .
  • LightMix Modular assays Roche for envelope protein E , RNA-dependent RNA polymerase RdRP , and nucleocapsid protein N genes multiplexed with EAV, the Real-Time Fluorescent RT-PCR kit for detecting 2019-nCoV BGI Biotechnology, Wuhan, China, and the TaqPath COVID-19 Combo Kit Thermo Fisher Scientific were also tested according to the manufacturers՚instructions.
  • Reactions performed using a Light Cycler 480 System II (Roche) , and threshold cycle (CT) values were determined by the second derivative maximum method, except for the CDC and TaqPath COVID-19 Combo Kit assays, which were performed using Applied Biosystems 7500 Fast or Quant Studio Real-Time PCR Systems (Thermo Fisher Scientific) using a fixed threshold of 0.1.
  • Loop-mediated isothermal amplification (LAMP) assay was performed using a Loopamp SARS-CoV-2 detection kit LMP403 and LoopampEXIA real-time turbidimeter

Mayank