Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

Swab samples should be collected using Dacron or Polyester swabs. Other specimen types should be transferred in sterile containers. In the transport phase, Viral Transport Medium (VTM) (Preperation of viral transport medium.

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Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

Table 1. Kit content [Shelf Life: 18 months; refer to the expiration date on the box. Each reagent stored at storage temperature, may be used until the expiration date indicated on the tube. The expiration date of the kit is determined by the expiration date of the reagents].

Storage Temperature: -20°C, Transport Temperature: +2-8°C
Content/Intended UseContentQuantity (10 p.L Reactions)Consumptio n / Reaction
100 Rxns250 Rxns500 Rxns1000 Rxns
SARS-CoV-2 (RdRp), SARS-CoV-2 (N) and

SARS-CoV-2 (ORF1ab) genes (FAM)

CVD Tri Oligo Mix1 x 250 uL1 x 625 uL1 x 1250 uL2 x 1250 uL2.5 uL
Internal Control (IC) (RNase P) (HEX)
DNA polymerase, dNTP mix, reaction buffer, reverse transcriptase and ribonuclease inhibitor2X Prime Script Mix1 x 500 uL1 x 1250 uL2 x 1250 uL4 x 1250 uL5 uL
Storage Temperature: +2-8°C/-20°C; Transport Temperature +2-8°C/-20°C

If the components are frozen store at -20°C, it should be stored at 2-8 °C without being frozen again after thawing.

Negative Control Template

Test it in each run for contamination control

NTC1 x 1000 uL1 x 1000 uL1 x 1000 uL1 x 1000 uL2.5 uL
Positive Control Template: Synthetic SARS- CoV-2 and RnaseP genom fragment

Test it in each run for reactive stability control

PC CVD Tri1 x 250 uL1 x 250 uL1 x 500 uL2 x 500 uL2.5 uL

Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

Intended Use and Test Principle

Kit is used for detecting the epidemic virus SARS-CoV-2 causing Coronavirus Disease 2019 (COVID-19). The kit allows to achieve RT-qPCR result in less than 50 minutes. The kit is applied to nucleic acid extracts obtained with vNAT extraction buffer or robotic extraction systems from nasopharyngeal aspirate/lavage, bronchoalveolar lavage, nasopharyngeal swab, oropharyngeal swab and sputum samples.

Rapid diagnosis with the kit is achieved via one-step reverse transcription (RT) and real-time PCR (qPCR) (RT-qPCR) targeting SARS-CoV-2 specific N and ORF1ab genes fragment. The oligonucleotide set targeting human RNase P gene functions as a control of the sampling, nucleic acid extraction and inhibition.

The kit is validated with RINATM M14 Nucleic Acid Extraction System (Robot Cat No: RINA-M14-01; Consumables Cat No: RN- NA-14-111-100) and vNAT Extraction Consumables (vNAT Viral Nucleic Acid Buffer Cat No: BS-NA-510 ; vNAT Transfer Tube Cat No: BS-NA-513-100). The kit is validated for 10 and 20 ^L qPCR volumes using Roche LightCycler® 96, Bio-Rad CFX96 TouchTM, Bio Molecular Systems Magnetic Induction Cycler (MIC), Qiagen Rotor-Gene® 5 Plex Real-Time PCR systems. The LOD of the kit is 5.6 SARS CoV-2 genome / reaction for all kit versions. The exclusivity tests of the kit were tested both in the laboratory and

in-silico using a pool sample prepared with 40 different viral and bacterial strains and nasal washing fluids obtained from 20 different people. The kit does not cross-react with other respiratory pathogens and human respiratory microbial flora. In-silico tests have shown that the kit cross-reacts with some bat-associated SARS-CoV strains.

Sensitivity and specificity of the kit; Tested on 357 positive and 94 negative clinical specimens archived according to the FDA approved RT-qPCR assay kit targeting SARS-CoV-2. The relative sensitivity and specificity of the kit were determined to be 97.3% and 100% for all kit versions.

Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

Collection, Storage and Shipment of Clinical Specimens

Swab samples should be collected using Dacron or Polyester swabs. Other specimen types should be transferred in sterile containers. In the transport phase, Viral Transport Medium (VTM) (Preperation of viral transport medium, Center for Disease Control and Prevention, SOP#: DSR-052-01) or Bio-Speedy® vNAT Viral Transfer Tubes (Cat No:BS-NA-513-100). Samples should be stored and transported at 2-8 °C until they arrive at the laboratory. Swab samples should be transferred within 5 days, other sample types should be transferred within 2 days. If a delay in shipment is expected, samples should be frozen at -70°C and shipped with dry ice. It is important that samples are not exposed to continuous freeze-thaw exposure.

Warnings

  1. The kit should be stored away from nucleic acid sources and qPCR amplicons.
  2. The components in the kit should not be mixed with components with different lot numbers or chemicals of the same name but from different manufacturers.
  3. Master stock reagents should be kept on the cold block during the PCR setup; if possible, the PCR setup should be performed on the cold block.
  4. Kit components should be mixed by gently shaking before use.
  5. The micropipettes used for pipetting qPCR mixes and template nucleic acids should be separate.
  6. Template nucleic acid and positive control tubes should always be kept closed, except for fluid transfers.
  7. The wipeable surfaces of the rooms, benches and devices where the test is performed should be cleaned regularly with 10% NaClO.
  8. The qPCR completed reaction tubes should be disposed of before opening in the laboratory.

RT-qPCR Application Protocol

Before starting the assay, please consider the following:

  • The kit was validated only for the template nucleic acid volume that is 25% of the total qPCR volume.
  • The kit can not be used with real-time PCR instruments without the periodic maintenance records.
  • Both white and clear 0.1 mL qPCR plates can be used for the assay, while slightly better performance can be obtained using the white plates for Bio-Rad CFX 96 and Roche Light Cycler 96 instruments.
  • 1 ml and 0.2 ml clear qPCR tubes can be used for the assay, while slightly better performance can be obtained using the 0.1 ml tubes for Rotor Gene Q instrument.
  • Device-specific reaction strips are used in the BMS MIC qPCR device.

Program the qPCR device as follows and add the reagents to the qPCR tubes in the order specified below, close the tubes, place them into the qPCR device and start the run (Table 2).

Table 2. Reaction set-up and qPCR program details

Reaction setupqPCR Program
ComponentReactionCycle NumberTemperatureDuration
2X Prime Script Mix5 pL10 pL152 °C5 min
Oligo Mix2.5 pL5 pL195 °C10 sec
4095 °C1 sec
Template Nucleic Acid2.5 pL5 pL55 °C1 sec
TOTAL REACTION VOLUME10 pL20 pLFAM/HEX/ROX/Cy5 Read* (Green/Yellow/Orange/Red Read)

Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

Interpretation of The Assay Results

The recommended threshold levels to calculate the number of threshold cycles (Cq) for both 10 ^L and 20 ^L reactions are 0.05 and 200 RFU for Roche LightCycler® 96, Bio-Rad CFX96 Touch™ respectively. In Rotor-Gene® instruments, the sigmoidality of the amplification curves should be evaluated from the “Raw Data” screen. To see the Ct values of sigmoidal curves in Rotor­Gene® devices; on the analysis screen, “Dynamic Tube” should be active, “Slope Correct” options should be passive, “Outlier Removal” option should be “0”, the threshold level should be set to 0.02. For BMS MIC qPCR, “Non-Assay Green/Parameters/Fixed Length” options should be selected, auto-threshold setting should be active.

Shape of the amplification curves obtained in the FAM/HEX/ROX/Cy5 (Green / Yellow / Orange / Red) channels are examined and non- sigmoidal curves are recorded as negative. The result is recorded as negative if there is no sigmoidal curve. The result is recorded as positive if Cq<38 and the analysis result should be interpreted according to Table 3. If there is no sigmoidal curve in the negative control and the sample is Cq>38, the nucleic acid extract from the sample should be tested again, and if Cq>38 is in the second analysis, a sample should be requested from this patient again and the analysis should be repeated.

Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

Table 3. Interpretation of Patient Samples

CasesNA IsolatePositive ControlNegative ControlComment
TargetICTargetICTargetIC
Case 1PosPosPosPosNegNegTarget Positive
Case 2PosPosNegNegNegNegTarget Positive: Since the sample gives a positive result, it is concluded that the kit works, the patient result is reported.If the problem persists in the positive control, contact the manufacturer and request a new positive control.
Case 3PosNegPosPosNegNegTarget Positive
Case 4NegPosPosPosNegNegTarget Negative
Case 5PosPosPosPosPosNegContamination Problem: The experiment is repeated by paying attention to the issues in the warnings section.
Case 6NegPosPosPosPosNegTarget Negative: Since the target is negative, there is no contamination problem. NTC supplied with the kit contents may be contaminated. If the problem persists, the manufacturer is contacted and a new negative control is requested.
Case 7NegNegPosPosNegNegSampling / Extraction / Inhibition Problem: Nucleic acid isolate is diluted 1/10 and the experiment is repeated. If the diluted sample does not produce positive result in the HEX channel, a new sample is requested.
Case 8NegNegNegNegNegNegReagent Problem: Reagents are renewed by contacting the manufacturer and the reaction is repeated.
Case 9NegPosPosPosNegPosIf the IC Cq value in the negative control reaction is 3 cycles or greater than the IC Cq value obtained from the NA isolate, the result is evaluated and reported as negative. Otherwise, a sampling / extraction / inhibition problem is concluded.
Case 10PosPosPosPosNegPosTarget Positive: Since the target is negative in the negative c ontrol and the target is positive in the NA isolate, the sample is reported as positive regardless of the contamination in the IC.

Bio-speedy® SARS CoV-2 Triple Gene RT-qPCR

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