Lightmix sarbecov e gene

Manufactured by TIB Molbiol

Sourced in Germany

The LightMix® SarbecoV E-gene is a real-time PCR assay for the detection of the E-gene of Sarbeco-like viruses, including SARS-CoV-2. The assay is designed to amplify and detect a specific region of the E-gene, which is a highly conserved genetic target among Sarbeco-like viruses.

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Lab products found in correlation

Cobas 6800, by Roche (2 mentions) Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions) Agpath idtm one step rt pcr reagents, by Thermo Fisher Scientific (1 mentions) Eav control, by TIB Molbiol (1 mentions) Lightcycler 480, by Roche (1 mentions) Rdrp gene, by TIB Molbiol (1 mentions) Architect sars cov 2 igg, by Abbott (1 mentions) Alinity m, by Abbott (1 mentions) Cobas sars cov 2 test kit, by Roche (1 mentions) Panther fusion, by Hologic (1 mentions) Xpert xpress sars cov 2, by Cepheid (1 mentions) Realstar sars cov 2 rt pcr kit 1, by Altona Diagnostics (1 mentions) Cobas sars cov 2 kit, by Roche (1 mentions) Lightcycler 480 2, by Roche (1 mentions)

Spelling variants of this product

LightMix® SarbecoV E-gene kit (5 citations) LightMix® SarbecoV E-gene plus EAV control (3 citations) LightMix SarbecoV E-gene assay (2 citations)

6 protocols using lightmix sarbecov e gene

SARS-CoV-2 Detection Protocols Comparison

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(Assay I) Nucleic acid extraction was performed using the MagNAPure® 96 DNA and Viral NA Kit, followed by amplification on LightCycler® 480II (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Detection of SARS-CoV-2 was conducted using the RealStar® SARS-CoV-2 RT-PCR Kit1.0 (altona Diagnostics, Hamburg, Germany) or LightMix®SarbecoV E-gene plus equine arteritis virus (EAV) control (TibMolBiol, Berlin, Germany). (Assay II) Processing of swabs was implemented with Panther Fusion® Hologic® and SARS-CoV-2 was detected using 5 μl of total RNA in 20 μl of LightMix®SarbecoV E-gene plus β-globin as internal control (TibMolBiol, Berlin, Germany). As second target the N-gene was amplified (inhouse primer sets in multiplex PCR, data unpublished). (Assay III) Detection of SARS-CoV-2 was performed on a Roche cobas®6800 using the cobas® SARS-CoV-2 kit targeting the E-gene/ORF-1a/b regions according to manufacturer's protocol.
SARS-CoV-2 was quantified using dilution series of cell culture supernatant extrapolated to approved standards (INSTAND e.V., Düsseldorf, Germany), measured in all assays, and Ct-values adjusted to Assay III (Ct_a).

Wunsch M., Aschemeier D., Heger E., Ehrentraut D., Krüger J., Hufbauer M., Syed A.S., Horemheb-Rubio G., Dewald F., Fish I., Schlotz M., Gruell H., Augustin M., Lehmann C., Kaiser R., Knops E., Silling S, & Klein F. (2021). Safe and effective pool testing for SARS-CoV-2 detection. Journal of Clinical Virology, 145, 105018.

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SARS-CoV-2 RT-qPCR Detection Protocol

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The 5 μl of the extracted RNA elute/sample was subjected to RT-qPCR for the qualitative detection of SARS-CoV-2 RNA utilizing with AgPath-IDTM One-Step RT-PCR Reagents (Thermo Fisher Scientific) using a Applied biosystem (ABI) 7500 Real Time PCR system (Thermo Fisher Scientific) and LightMix^® SarbecoV E-gene (TIB MOLBIOL). Reactions were heated to 55°C for 5 minutes for reverse transcription, denatured in 95°C for 5 minutes and then 45 cycles of amplification were carried out for 95°C for 5 seconds and 60°C for 15 seconds using FAM parameter for E gene. This assay targets the detection of E gene for SARS as well as nCoV-2. The primer details are given in Table 1. All samples that were screened positive for E gene were confirmed by performance of RT-qPCR for the detection of specific RdRP gene of SARS-CoV-2 using LightMix^® Modular SARS-CoV-2 RdRP (TIB MOLBIOL) using similar PCR conditions as described above.

Gupta E., Padhi A., Khodare A., Agarwal R., Ramachandran K., Mehta V., Kilikdar M., Dubey S., Kumar G, & Sarin S.K. (2020). Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: A reliable, faster and economical method. PLoS ONE, 15(7), e0236859.

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SARS-CoV-2 RT-PCR Detection Protocol

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Reverse transcriptase-polymerase chain reaction (RT-PCR) was used as the reference test. The RT-PCR samples were collected by health-care workers using nasopharyngeal swabs. At the study laboratory, the samples were initially lysed to inactivate any viral agent, followed by extraction of the viral RNA in separate biosafety class 2 cabinets. The next step involved the mastermix preparation and template addition in separate hoods. The final stage was amplification using AriaMx real-time PCR thermocycler. The reagent used was LightMix SarbecoV E-gene (manufacturer TIB MOLBIOL) which is the standard assay used in the Public Health Reference Laboratory. The assay targets only the E-gene of SARS-CoV-2. Further details and test properties can be found in the instructions for use [16 ]. A Ct value of <40 is interpreted as a positive and Ct values ≥40 as a negative test result for SARS-CoV-2.

Abdul-Mumin A., Abubakari A., Agbozo F., Abdul-Karim A., Nuertey B.D., Mumuni K., Heuschen A.K., Hennig L., Denkinger C.M., Müller O, & Jahn A. (2021). Field evaluation of specificity and sensitivity of a standard SARS-CoV-2 antigen rapid diagnostic test: A prospective study at a teaching hospital in Northern Ghana. PLOS Global Public Health, 1(12), e0000040.

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Quantifying SARS-CoV-2 Viral Load by qPCR

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Cycle threshold values for quantifying viral load in naso/oro-pharyngeal swabs was done by qPCR using LightMix® SarbecoV E-gene(Corman et al., 2020 (link)) plus EAV control (TIB Molbiol, Berlin, Germany) in combination with the N-gene (inhouse primer sets in multiplex PCR) on LightCycler® 480 (Roche Diagnostics).

Vanshylla K., Di Cristanziano V., Kleipass F., Dewald F., Schommers P., Gieselmann L., Gruell H., Schlotz M., Ercanoglu M.S., Stumpf R., Mayer P., Zehner M., Heger E., Johannis W., Horn C., Suárez I., Jung N., Salomon S., Eberhardt K.A., Gathof B., Fätkenheuer G., Pfeifer N., Eggeling R., Augustin M., Lehmann C, & Klein F. (2021). Kinetics and correlates of the neutralizing antibody response to SARS-CoV-2 infection in humans. Cell Host & Microbe, 29(6), 917-929.e4.

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Multimodal Approach for SARS-CoV-2 Identification

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SARS-CoV-2 infections were identified using a multimodal approach in which biomaterial was collected at baseline and follow-up. To identify acute infection, throat swabs were analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using the Light Mix SarbecoV E-gene (plus EAV control) and RdRP-gene (TIB Molbiol, Germany). The methods used for RT-qPCR have been described elsewhere (laboratory 2) [17 (link)]. EDTA plasma samples were analyzed for circulating antibodies to the SARS-CoV-2 nucleocapsid with a qualitative microparticle chemiluminescent immunoassay (Architect SARS-CoV-2 IgG, Abbott, Germany) with a threshold of 1.4 relative light units and second, a qualitative microparticle electro-chemiluminescence immunoassay (Elecsys Anti-SARS-CoV-2 Pan-Ig, Roche, Germany) with a cutoff index of 0.8. Any self-reported PCR results were additionally included which were collected retrospectively via a computer-assisted personal interview and continuously via a weekly smartphone-app survey. Individuals were considered infected if any of these measurements were positive. Detailed information on sample preprocessing and storage is provided in the Supplementary Appendix.

Baumkötter R., Yilmaz S., Zahn D., Fenzl K., Prochaska J.H., Rossmann H., Schmidtmann I., Schuster A.K., Beutel M.E., Lackner K.J., Münzel T, & Wild P.S. (2022). Protective behavior and SARS-CoV-2 infection risk in the population – Results from the Gutenberg COVID-19 study. BMC Public Health, 22, 1993.

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Comparative SARS-CoV-2 RT-qPCR Detection

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Different SARS-CoV-2 RT-qPCR protocols were used for detection of SARS-CoV-2. In brief, the following methods were used: (i) cobas® SARS-CoV-2 test kit running on the cobas 6800® (Roche Diagnostics, Mannheim, Germany) was used for 385 (55.32%) samples according to the manufacturer's instructions. (ii) SARS-CoV-2 AMP kit on the Alinity m (Abbott, Illinois, USA) was used for 62 (8.91%) samples. (iii) Multiplex RT-qPCR with LightMix® SarbecoV E-gene (TIB Molbiol, Berlin, Germany) running on the Panther Fusion® (Hologic, Wiesbaden, Germany) was used for 2 (0.29%) samples. (iv) For 244 (35.06%) specimens, samples from up to ten asymptomatic employees were pooled and tested for SARS-CoV-2 infection using the methods (i) and (ii). All samples within a negative pool were considered negative. For positive pools all samples were retested individually using the methods (i) and (ii). (v) Xpert® Xpress SARS-CoV-2 (Cepheid, Sunnyvale, USA) test kit was used for 3 (0.43%) samples according to the manufacturer's instructions. To enable comparison of Cycle threshold (Ct) values between different RT-qPCR protocols, Ct values were translated into copies/ml and converted to a cobas 6800®-adjusted Ct value. Detailed descriptions of the RT-qPCR protocols and Ct value conversion can be found in previous publications [10 , 27 ].

Poopalasingam N., Korenkov M., Ashurov A., Strobel J., Fish I., Hellmich M., Gruell H., Lehmann C., Heger E, & Klein F. (2022). Determining the reliability of rapid SARS-CoV-2 antigen detection in fully vaccinated individuals. Journal of Clinical Virology, 148, 105119.

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