EQ Flu A/B One Step RT qPCR kit for wastewater contains a 5x reaction buffer optimized for sensitive and specific detection of Influenza A virus and Influenza B virus RNA species from various sources and purity backgrounds. Enzymes come separately in 20x and 200x formulations to provide flexibility and allow user to optimize depending on the purity of the substrate of interest (mainly on the presence of PCR inhibitors). The 10x Flu A/B Oligos mix contains a combination of primers and probes optimized for the detection of Influenza A virus and Influenza B virus at either FAM or HEX channel respectively. Optimizations on wastewater extracts samples guarantee the reproducible and efficient qPCR reactions close to the expected limit of detection (<10 copies per reaction).

Reagents supplied:

Standard 20 μl reaction:

*0.08-0.12 μl may be added depending on the substrate used. For Reference Labs wastewater extracts analysis, 0.1 μl has been used extensively used. Do not add more than needed, because PCR inhibition effect may be caused by the excess of Reverse Transcriptase enzyme in the reaction.  

**3 μl of RNA extracts is recommended. In case of high concentration of PCR inhibitors, perform 1:10 dilution of the original wastewater extract sample and add 2-4 μL per reaction. 2 μL of wastewater extracts from Reference Labs have been extensively used in our analysis. 

Optional: Depending on the wastewater quality, the region or the season collected, treatment of wastewater extracts with PCR inhibitors removal kits like the ones provided by ZYMO Research (https://zymoresearch.eu/collections/onestep-pcr-inhibitor-removal-kits), may be important to obtain accurate, quantifiable results. In order to verify the necessity of this type of treatment, test 4 independently extracted samples, collected over 2-4 weeks, and compare the quantification of the 1:10 dilution of either treated or untreated sample. If the difference obtained is lower than 30 % then that treatment may be omitted from the analysis. 

One Step-RT recommended conditions for single and multiplex One Step RT-qPCR reactions.

Step 1: RT reaction. Important step to ensure success in detecting and quantifying efficiently low level transcripts. RT step may be increased further to 25 minutes to further enhance specificity. But it is not necessary in all cases. It is not recommended to compare levels between runs that have been set with different RT times. Test in the beginning and proceed with the same program each time to ensure quality comparisons.

Step 2: RT inactivation/ Hot Start Taq DNA polymerase activation.

Initial activation of Hot Start at 94^oC for 15 minutes is very important in the case of reactions with very low PCR template copies and for obtaining the maximum efficiency in qPCR setups.  DO NOT USE FEWER THAN 10 MIN!!

Step 3: Cycle denaturation.

Step 4: Annealing/Extension step. The recording is on FAM and HEX channel.

 After the analysis finishes, set the baseline in the area where maximum efficiency is obtained. For Bio-Rad platforms, set at 150-350 Relative fluorescent units (180 RFUs is our standard practice). For other platforms adjust according to your standard practice.

(Optional/ When applicable) Quantify samples with or without PCR inhibitor removal treatment and see whether there is a difference greater than 30 %. If there is, then the PCR inhibitor removal kit may be necessary before the RT-qPCR analysis for the accurate quantification of the viral load in the wastewater sample. If in both cases, treatment or not, a similar quantity is estimated for the same sample, then the PCR inhibitor removal step may be omitted.

Additives that improve robustness 

In cases of samples with low viral content near the limit of detection (~5-10 copies/μl of wastewater sample) and/or high concentration of inhibitors, one may also add extra qPCR enhancer. Try in steps of 0.01 μL.

Up to 0.12 μl / reactions have been shown to help in some cases. The quantity provided in each kit (24 μl) suffices for at least 200 reactions with 0.12 added. We strongly suggest testing the recommended quantity mentioned at Table 2 before increasing the amount per reaction.

Appendix 1 

 10⁵ to 10 copies of FluA and FluB IVT RNA. 3 μl were used as template. Result has been produced with kit RN017S, lot: RN017 0100. 

Other Quality Controls:

Certified as DNase-RNase free using fluorescently labeled RNase and DNase probes.  Efficiency between 90-110%, with R² value >0.99 of each lot is estimated by a standard curve assay produced over 5 logs IVT RNA substrate dilutions. Stock provided by waste-water surveillance lab of Thesaloniki and quantified at 8 x10⁶ cp/μL  for Flu A and 1.2 x 10⁷ cp/μL for Flu B targets.

Shipping

Shipped on blue ice or dry ice.

Storage conditions

Store at -20^οC ± 5^οC