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FAQs about the CDC DENV-1-4 Real-Time RT-PCR Multiplex Assay

Q1: Is the CDC DENV-1-4 rRT-PCR multiplex assay appropriate for screening mosquitoes or for detecting dengue virus in animals?

Answer: The assay has been cleared by the US Food and Drug Administration (FDA) for diagnosis of dengue in humans only.

Q2: Are the oligonucleotide sequences published or available?

Answer: The sequences for the oligonucleotides along with all characteristics and performance evaluations of the assay have been published.

Q3: We would like to use the CDC DENV-1-4 rRT-PCR multiplex assay on plasma from patients with suspected acute malaria and recent travel in our region. Is this acceptable?

Answer: The assay is 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 the malaria smear or rapid diagnostic test (RDT) is negative. In that case, dengue would be part of the differential diagnosis.

Q4: Where can I find instructions for testing the proficiency panel (PT)?

Answer: The PT panel is to be tested using the package insert included with the assay. Extraction volumes are specified by manufacturers of the RNA extraction methods approved for use with the CDC DENV-1-4 rRT-PCR multiplex assay. Extract only one replica of each panel.

Q5: We usually test in duplicate and repeat positive samples with a second extraction. Our state regulations specify that proficiency test samples should be tested in the same manner as patient samples. Should we perform this panel with identical procedures? What are the preferred extraction loading and elution volumes?

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 standard operating procedure requirements. Our test has been validated to ensure that threshold cycles (CTs) ≤ 37.00 are near 100% reproducible.

Q6: When running the CDC DENV-1-4 rRT-PCR multiplex assay, the non-template control (NTC) and human specimen controls (HSC) are crossing the threshold for DENV-2 primer/probe set with CT values greater than 38. Is this of concern?

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, rRT-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

Q7: Should the positive control RNA be diluted 1:10 after it is extracted? The protocol indicates this on page 7, but not in the instructions on page 14.

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.

Q8: In an effort to validate the CDC DENV-1-4 rRT-PCR multiplex assay, we ran serial dilutions of the DENV-1-4 Control Mix. Why are there discrepant results between the singleplex and multiplex assays?

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 (

Q9: Do whole blood specimens have the same shipping requirements as serum/plasma?

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. CDC prefers 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.

Q10: One tube of the DENV-1-4 positive control mix of human specimen controls (HSC) is adequate for approximately 7 extractions. Can it be thawed 7 times or should it be aliquoted after the first thaw to prevent multiple freeze-thaw cycles? Does the HSC have a limit of 5 freeze-thaw cycles similar to the CDC flu assay?

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).

Q11: What are the limits of detection for each serotype?

Answer: The limit of detection at 100% reproducibility is approximately 2-3 x103 genome copies equivalents per mL (GCE/mL) for all serotypes. Dilutions below 102 GCE/mL can be detected but not with 100% reproducibility.

Q12: Can the CDC DENV-1-4 rRT-PCR multiplex assay be run on other instruments different from the ABI 7500Dx?

Answer: The assay is FDA cleared 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.

Q13: How much does the kit cost?

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 You will be asked to provide information about your intended use of the assay.

Q14: What is the difference between the US and international versions of CDC DENV-1-4 rRT-PCR multiplex assay kits?

Answer: The US kit contains suspended DENV-1-4 control mix and human specimen controls. The international kit contains the same controls, lyophilized. These controls should 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 United States.

Q15: Regarding the human specimen controls (HSC) and positive control extraction, page 17 has a disclaimer to follow manufacturers’ recommended procedures for sample extraction. However, the CDC protocol states that final volume of eluted RNA should equal the volume of extracted material. What volumes should we use?

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 70 uL.

Q16: What does CDC recommend for assay validation? Can I get more positive test material to complete the verification of replicates?

Answer: The CDC can provide additional samples for validations depending on your requirements. Please contact our support group at

Q17: What is the average CT of the HSC when tested in CDC’s laboratory on the CDC DENV-1-4 rRT-PCR multiplex assay?

Answer: Negative for dengue and positive for ribonuclease P (RP) (CT around 30).

Q18: The package insert says to reference PCR negative samples to dengue IgM. Are these performance testing samples acceptable for serologic testing? Can I use them to validate our serologic test?

Answer: The performance testing samples and the positive controls distributed with the CDC DENV-1-4 rRT-PCR mulitplex assay kit contain DENV-1-4 RNA and not antibodies. These samples are for use with the kit and should not be used for serological testing, such as for IgM  antibody. The rRT-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 rRT-PCR multiplex assay is recommended for use during the first 7 days after symptom onset (allowed by FDA to be used until day 7). Samples collected after 4 days of symptoms or negative on the CDC DENV-1-4 rRT-PCR multiplex assay should be tested by an approved enzyme immunoassay (EIA) for IgM anti-DENV.

For more information on EIAs for dengue diagnostic testing, visit CDC’s dengue laboratory guidelines where you can obtain contact information and request more help for EIA proficiency testing.