Xfree™ COVID-19 DIRECT RT-PCR
Is the Xfree™ COVID-19 Direct RT-PCR test affected by the new mutations in the SARS-CoV-2 genome initially identified in the UK, South Africa, and India?
Delta Variant (B.1.617.2) Update – 14 July 2021
There are 17 non-synonymous mutations and deletions identified to date within the B.1.1.7 lineage, with 15 of those being located in the ORF1ab, ORF8, and Spike Protein genes. None of these mutations affect viral detection ability by any of the BioGX assays which target the N gene. Moreover, out of the 17 identified mutations only 2 are located in the N gene region, but are located outside of the region targeted by BioGX assays. The BioGX assays target a unique region of the N gene which is unaffected by the N gene mutations in the B.1.1.7 lineage or the 501Y.V2 variants.
BioGX performed exhaustive genomic sequence database (GISAID & NIH) analysis in order to determine if any of the genomic sequences from the potentially more infectious B.1.1.7 lineage or 501Y.V2 variant possess any mutations in the N region targeted by the BioGX test might affect detection of the virus. Our in silico analysis utilized over 4,200 SARS-CoV-2 B.1.1.7 lineage genomes available from the GISAID and NIH databases (as of January 4, 2021).The predicted detection of 99.9% of the SARS-CoV-2 B.1.1.7 lineage remains unchanged with the BioGX tests. In silico analysis of the 320 sequences available in the GISAID database (as of January 4, 2021) belonging to the 501Y.V2 variant (within B.1.351 lineage) predicted no change in PCR detection with the BioGX tests.
BioGX COVID-19 Related Assays
BioGX utilizes primers and probes for detection of SARS-CoV-2, targeting the viral nucleocapsid gene (N gene region), human RNaseP gene as an endogenous control, and a non-naturally occurring internal amplification control (IAC). Both the SARS-CoV-2 N1/N2, and RNase P primer/probe sets are based upon those designed and recommended by the US Centers for Disease Control and Prevention.
The following four BioGX SARS-CoV-2 testing products utilize the US Centers for Disease Control and Prevention primer/probe set designs for N-gene region(s):
- BioGX Xfree™ COVID-19 Direct RT-PCR
- BD BioGX SARS-CoV-2 Reagents for BD MAX™ System
- BioGX SARS-CoV-2 HMP – N1, N2 & RNase P Multiplex
- BioGX COVID-19, Flu A, Flu B, RSV RT-PCR for BD MAX™
- Xfree™ SARS-CoV-2-XMP Extraction-Free Direct PCR -Multi-Platform – SARS-CoV-2 N1, RNase P, IAC
- BioGX SARS-CoV-2 N1, N2, E-gene, RNase P/Flu A, Flu B, RSV A/B – Multi-Platform
In summary, as of July 14, 2021, in silico analysis of the N gene regions targeted by the BioGX tests predicts strains within the potentially more infectious B.1.617.2, B.1.1.7 lineage and 501Y.V2 variants (within B.1.351 lineage) of SARS-CoV-2 circulating in the United Kingdom, South Africa, India, and other countries will be detected.
I am currently utilizing SARS-CoV-2 diagnostic tests that identify SARS-CoV-2 via two or three diagnostics targets. Since the BioGX Xfree™ COVID-19 Direct RT-PCR test uses only one SARS-CoV-2 diagnostic target (N1 gene region), is this a limitation in virus identification? If not, what is the status of the BioGX Xfree™ COVID-19 Direct RT-PCR EUA review?
The FDA guidelines provided in June 2020 supported the use of single target SARS-CoV-2 identification as an acceptable approach for clinically validated tests. The FDA has granted EUA to the BioGX Xfree™ COVID-19 Direct RT-PCR test. (https://www.fda.gov/media/
The FDA lists the BioGX Xfree™ COVID-19 Direct RT-PCR product under the Question: What commercial manufacturers are distributing diagnostic test kits under the policy outlined in Section IV.C of the Policy for Coronavirus Disease-2019 Tests? (Updated 12/14/20). (https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/notifications-and-emergency-use-authorizations-faqs-testing-sars-cov-2).
Xfree™ Related Questions
What is the BioGX Xfree™ COVID-19 Direct RT-PCR test?
The BioGX Xfree™ COVID-19 Direct RT-PCR (Xfree COVID-19) is a real-time reverse transcription (RT) polymerase chain reaction (PCR) test provided as a complete lyophilized RT-PCR master mix (Sample-Ready™) capable of detecting the novel SARS-CoV-2 RNA presence by direct patient sample addition to the master mix. For direct sample addition, there is no pre-processing of the sample such as heat or chemical lysis, dilution, or silica column-based or magnetic bead-based nucleic acid extraction procedure.
What gene region is targeted by the assay?
The test targets the N1 region of the N gene of SARS-CoV-2. The SARS-CoV-2 and the RNase P primers and probes included in the test are identical to the sequences used by the US Centers for Disease Control and Prevention in CDC’s FDA-EUA method.
What are the advantages of the BioGX Xfree™ COVID-19 test?
Fast turnaround time without expensive automation, ease of use, and improved throughput are key advantages of the BioGX Xfree COVID-19 test. The robust chemistry of the test allows addition of 5µL of the patient sample directly to 15µL of the RT-PCR mix. There is no pre-processing of the sample required. Depending on the real-time PCR instrument used, 96 or 384 sample results can be obtained in less than 80 mins.
What real time PCR instrument is required for this test?
The BioGX Xfree™ COVID-19 Direct RT-PCR test is validated for Applied Biosystems™ 7500 Fast Dx, Applied Biosystems QuantStudio™ 5, the Bio-Rad CFX96 Touch™ and Bio-Rad CFX384 Touch™ real-time PCR instrument.
Can I use the BioGX Xfree™ COVID-19 test with other qPCR instruments?
The use of other qPCR instruments is considered a modification and the modified test should be validated by using a bridging study to the EUA test. Please review FDA-EUA policy for further guidance on using alternate instruments not validated by the test manufacturer.
Do I need to add any other reagents to the PCR reaction after rehydrating the lyophilized master mix with water?
No, the BioGX Xfree COVID-19 reagent is a complete (Sample-Ready™) master mix that only requires rehydration with molecular grade water, aliquoting 15µL to the PCR well, and then direct addition of 5µL of patient sample, sealing of the reaction tube/plate, and running on the recommended real time PCR instrument.
How many reactions can I get out of a single lyophilized tube?
BioGX guarantees 26 (extraction free workflow) or 40 reactions (extracted samples workflow) after a single lyophilized tube is rehydrated with 400uL of Nuclease-free water. For direct sample testing, 15µL of the master mix is aliquoted into the PCR well and 5 µL of sample is directly added. When using extracted samples, only 10µL of the rehydrated master mix is used with 5µL of the eluted nucleic acid from the extraction method.
Is there a viral inactivation step when using the BioGX Xfree™ COVID-19 Direct RT-PCR test?
All patient samples and viral controls should be handled by following proper Biosafety Level II processes and procedures in accordance with state, local, and federal regulations. Patient samples and sample additions should be handled within a Class II (or higher) biological safety cabinet (BSC). When using the direct sample addition method, once all samples are added to the PCR plate/tube, the plate/tube is immediately sealed. The RT-PCR temperature cycling protocol includes a total hold time at 95°C for 7 minutes which is adequate to inactivate the SARS, if present in the sample. However, an additional viral heat inactivation step (95°C for 10 min) may be included at the end of the PCR cycle to ensure the virus is completely inactivated.
What specimen types have been validated with the test?
The test has been validated for sample types of Nasopharyngeal swab, Nasal swab, Mid-turbinate swab, Oropharyngeal swab transported in Copan ESwab™, Copan UTM®, BD UVT, VTM and dry swab (resuspended in saline) for both direct sample and nucleic acid extracted sample workflows.
Can I use the BioGX Xfree™ COVID-19 test for patient samples that have undergone a nucleic acid extraction procedure?
Yes, in addition to extraction-free processing the test is able to utilize extracted samples when extracted with a validated magnetic bead or silica column-based total nucleic acid extraction procedure.
Can I use more than the recommended direct sample patient volume in the reaction?
No more than 5 µL of patient sample should be used. The BioGX Xfree COVID-19 Direct RT-PCR test has been validated using a final reaction volume of 20 µL, consisting of 15 µL of master mix and 5 µL of patient matrix. Crude clinical specimen matrices contain substances that inhibit PCR and the use of more than 5 µL of the specimen is not recommended.
How does the test provide assurance that clinical specimens are successfully amplified and detected?
The BioGX Xfree™ COVID-19 Direct RT-PCR incorporates and detects a synthetic non-naturally occurring RNA sequence which is included and targeted as an Internal Amplification Control (IAC) to show the reaction conditions were appropriate for reverse transcription of RNA, real-time PCR amplification and detection. The assay also includes the human RNase P gene (RP), as an endogenous sample processing control (SPC) that ensures sufficient patient sample was collected and added to the RT-PCR reaction.
How can I identify inhibitory specimens when using the BioGX Xfree™ COVID-19 Direct RT-PCR test?
The BioGX Xfree™ COVID-19 Direct RT-PCR is a very robust mixture which allows direct sample addition. However, if a sample is analyzed that contains sufficient inhibitory substances, these samples can be identified by monitoring of the assay controls, the Internal Amplification Control (IAC), and the RNase P (SPC) performance. Samples for which the RNA IAC does not report, and are negative for N1, are considered indeterminate potentially due to excessive RT-PCR inhibition and should undergo RNA extraction using a validated magnetic bead-based or silica column-based nucleic acid extraction procedure to remove excessive inhibitors. Samples for which the RNase P or N1 target do not amplify within prescribed range are considered indeterminate due to inadequate amplifiable human nucleic acid. Please refer to IFU Table 2 (Interpretation of Patient Sample Results).
What if the sample is still inhibitory or fails to amplify RNase P after using a nucleic acid extraction method?
The sample is considered indeterminate, and BioGX recommends collecting a fresh sample and re-testing.
I am detecting the IAC with late Ct values when using direct ESwab™ in the PCR reaction? Does it mean that I have an inhibitory sample?
Several studies have demonstrated that ESwab™ collection device is a suitable alternative to the UTM®/VTM collection system in the context of an international swab shortage. In our studies, we have demonstrated that the use of pure liquid amies from the ESwab™ collection (no swab specimen) will delay the IAC amplification, so a later Ct for IAC is expected when using ESwab™ as a direct input into the PCR reaction. However, the use of direct ESwab™ will not affect the sensitivity of the test as this collection device has been validated as having robust N1 target detection in ESwab™ with inactivated virus. Please refer to Table 2 (Interpretation of Patient Sample Results) in the IFU.