The COVID-19 Surveillance Test Kit and Coronavirus Environmental Test Kit are both RT-qPCR tests for the detection of SARS-CoV-2. Test kit materials cannot be interchanged due to several key differences between the kits.
1. The internal control target in each kit is different.
Both kits detect a region of the SARS-CoV-2 N gene in the FAM channel of a qPCR instrument. However, the second target detected in the HEX channel is different between the two kits.
The second target in the Coronavirus Environmental Test Kit is an internal control that is included as a part of the Coronavirus Environmental Oligo Mix, one of the three components making up the Common Mix. This internal control is used to identify potential PCR inhibition and errors in PCR reaction setup to prevent false negatives. Since the Common Mix is added to every PCR tube, this target should amplify in every PCR reaction tube, including the No Template Control (DNase/RNase-Free Water).
In the COVID-19 Surveillance Test Kit, the second target is human housekeeping gene RPLP0, or ribosomal protein lateral stalk subunit P0. This gene encodes a ribosomal protein that is present in human specimens such as saliva. RPLP0 functions as an endogenous internal control and confirms successful RNA extraction and RT-qPCR for human specimens. Since the No Template Control is DNase/RNAse-Free Water rather than a human specimen, this target should not amplify in this PCR reaction tube.
2. The RNA extraction process is different.
SARS-CoV-2 RNA is much more difficult to extract from biological samples such as saliva than hard surfaces. Because of this, different extraction mechanisms are used for each sample type.
The Coronavirus Environmental Test Kit uses the One-Step DNA/RNA Extraction Buffer to extract SARS-CoV-2 from environmental surface swabs. After swabbing a hard surface, an environmental swab is inserted into a 1.5 mL tube containing 200 μL of One-Step DNA/RNA Extraction Buffer 1X, which is stable at room temperature. The swab is rotated ten times to release any viral material, then removed from the tube. The extracted SARS-CoV-2 RNA is now in your 1.5 mL tube with the buffer and is available for PCR.
The COVID-19 Surveillance Test Kit uses the Enzymatic DNA/RNA Extraction Buffer 10X which lyses saliva specimens via chemical and enzymatic processes. Buffer is added to the 1.5 mL tube containing the saliva specimens and incubated at room temperature for 15 minutes. During the extraction process, the enzymatic properties of the buffer inactivate DNases, RNases, and antimicrobial proteins, which stabilizes the extracted SARS-CoV-2 RNA. These enzymes are then inactivated before preparing the PCR reactions to avoid degradation of the Common Mix components, which occurs during a 10 minute, 95 °C heat incubation step. After a brief centrifugation, the sample lysate is ready for PCR.
While not necessary for adequate sensitivity, the Enzymatic DNA/RNA Extraction Buffer can be used with the Coronavirus Environmental Test Kit to improve the yield of RNA from environmental swab extractions. To do this, substitute 200 μL of the One-Step DNA/RNA Extraction Buffer 1X for 200 μL of the Enzymatic DNA/RNA Extraction Buffer 1X, then proceed with the RNA extraction as outlined in the product manual. Note that the 1X (rather than 10X) concentration of the Enzymatic DNA/RNA Extraction Buffer should be used for the extraction.
The One-Step DNA/RNA Extraction Buffer should not be used to extract SARS-CoV-2 RNA from biological specimens processed with the COVID-19 Surveillance Test Kit.
3. The Common Mix is different.
The process for preparing a Common Mix for the kits is similar: Sahara One-Step RT-qPCR Master Mix with UNG, an Oligo Mix, and a Cofactor Buffer are combined into a 1.5 mL tube. The Common Mix is then added to each PCR tube along with a sample lysate or control to facilitate each reaction.
However, the formulation of the Oligo Mixes and concentration of the Cofactor Buffers are different between the two kits. The Coronavirus Environmental Oligo Mix includes the chemistry required for amplifying and detecting the presence of the internal control, while the COVID-19 Surveillance Oligo Mix includes the chemistry required for amplifying and detecting the presence of the endogenous control.
Additionally, different ratios of Common Mix and sample lysate or controls are required for the PCR reaction. The Coronavirus Environmental Test Kit requires 15 μL of Common Mix and 5 μL of sample lysate or control, while the COVID-19 Surveillance Test Kit requires 17.5 μL of Common Mix and 2.5 μL of sample lysate or control for the highest degree of sensitivity.