FAQs about the CDC DENV-1-4 RT-PCR Assay
Answer: The CDC DENV-1-4 RT-PCR Assay has been approved by the FDA for diagnosis of dengue in humans only.
Answer: CDC has not published the sequences used in the assay yet. CDC is currently working on a publication that will include the primer and probe design.
Answer: The CDC DENV-1-4 RT-PCR Assay is to be used for diagnosis of dengue on serum or plasma samples from dengue-suspected cases. Since patients with suspected malaria present an acute febrile illness similar to suspected dengue patients, they may be candidates for dengue diagnostic testing if they are malaria smear or RDT negative. In that case, dengue would be part of the differential diagnosis.
Answer: The PT panel is to be tested using the package insert of the CDC DENV-1-4 Real Time RT-PCR Assay. Extraction volumes are specified by manufacturers of the RNA extraction methods approved for the CDC DENV-1-4 RT-PCR Assay. Extract only one replica of each panel.
Answer: The proficiency panel does not need to be run in duplicates in order to understand how the test worked for your lab. This proficiency panel is not meant to fulfill the requirements of your state. For clinical samples, please follow your state or SOP requirements. Our test has been validated to ensure that CTs ≤ 37.00 are 100% reproducible.
Answer: Samples with a CT value of 37.01 or greater are considered negative. Only the NTC (not the HSC) should be run with dengue primers. NTC should not give background signal for any serotypes. However, real-time RT-PCR assays, especially multiplex assays with several primers and probes in one reaction, may sometimes give a slight signal very late in the test. We recommend that for every run, you analyze your results in “linear view”. This will allow you to set your threshold at the start of the exponential curve of the positive control mix for each serotype. You can then change to log view to analyze all results. The baseline should not be adjusted; it should be kept at 3 to 15. If you persistently detect a late signal for the NTC with CT values lower than 39, please contact the dengue PCR support group at denguePCRsupport@cdc.gov.
Answer: You can dilute the extracted RNA from the control mix, but CDC recommends that you run the control mix without a dilution in each run. The undiluted RNA should give you CT values of 25-30 and the 1:10 diluted RNA should give you CT values of 30-35.
Answer: The control mix is calibrated to give users a mid-positive result for all serotypes; however, the stoichiometry of the four DENV serotypes is not precisely 1:1:1:1. As the control mix is diluted, different serotypes will out-compete the others in the multiplex assay. Limits of detection and reproducibility of the assay should be determined by running the multiplex or singleplex assays on each DENV serotype template separately. The CDC can provide these templates upon request (denguePCRsupport@cdc.gov).
Answer: Whole blood should never be frozen since the red cells will hemolyze. If whole blood is collected, it should be centrifuged to separate it into serum or plasma. We favor dry-ice shipment of serum or plasma over use of cold-packs especially if transit will be longer than 24 hours. If whole blood cannot be centrifuged and separated into serum or plasma, it should be shipped at 4°C (cold-packs) but some hemolysis is likely unless a serum separator tube (tiger–top) is used.
Answer: CDC recommends minimizing the freeze-thaws on samples and controls CDC provides 4 replicas of these controls to avoid having to manipulate or freeze-thaw these controls unnecessarily. Aliquoting the controls after the first use is a good way to prevent excessive freeze thaws. It is also recommended that thawed samples and controls be kept on ice during processing (page 12, section 8.3 of the package insert).
Answer: The limit of detection at 100% reproducibility is approximately 103-104 genome copies equivalents per mL (GCE/mL) for all serotypes. Dilutions below 102 GCE/mL can be detected but not with 100% reproducibility.
Answer: The assay is FDA approved to be run on the ABI 7500Dx only in the United States. It would need to be optimized for the different PCR testing platforms for laboratories not using an ABI 7500Dx machine. The assay is serotype-specific and multiplexed, so it needs to be run on a cycler with the capacity to detect the 4 different specified Taqman labels or fluorophores.
Answer: Currently, the assay kit (primers, probes, and controls) is being distributed to public health laboratories free of charge. If you are interested in obtaining the assay kit, please contact denguePCRordering@cdc.gov. You will be asked to provide information about your intended use of the assay.
Answer: The US kit contains suspended DENV-1-4 control mix and human specimen controls. The international version of the kit contains the same controls, lyophilized. These controls are to be rehydrated in 1 ml of PCR-grade water. The two kits have different catalogue numbers (US: KK00128 and International: KK00129). KK00129 is not distributed in the US.
Answer: The package insert has been corrected with approval from the FDA. These changes include a correction on 10.2 and 10.3 on page 14. CDC eliminated the instructions where the HSC and positive control extraction and elution volumes should be maintained equal. The human control and the dengue virus control mix should be run in a manner consistent with the serum samples (or the proficiency panel samples) according to manufacturers’ specifications. That is, 140 uL of the sample and elute in 60 uL.
Answer: Negative for dengue and positive for RP (CT around 30).
Answer: The PT samples and the positive controls distributed with the CDC DENV-1-4 RT-PCR assay kit contain DENV-1-4 RNA and not antibodies. These samples are for RT-PCR testing with the kit and should not be used for enzyme immunoassays (EIA) testing such as for IgM anti-DENV. The RT-PCR negative samples referred to in the package insert are patient samples which may or may not contain antibodies to DENV. The CDC DENV-1-4 RT-PCR Assay is recommended to be used during the first 7 days after symptom onset. Samples collected after 4 days of symptoms or negative on the RT-PCR assay should be tested by an approved EIA for IgM anti-DENV.
For more information on immunoassays for dengue diagnostic testing visit CDC’s dengue laboratory guidelines page at https://www.cdc.gov/dengue/clinicalLab/laboratory.html where you can obtain contact information and request more help for your EIA proficiency testing.