Co-Dx VZDC-R-001 Vector Smart Zika or Dengue and Chikungunya Instruction Manual

Co-Dx VZDC-R-001 Vector Smart Zika or Dengue and Chikungunya

Vector Smart™ Zika, Dengue, and Chikungunya RUO CO-DIAGNOSTICS, INC.

Manufacturer:
Co-Diagnostics, Inc 2401 S Foothill Dr. Ste D Salt Lake City, UT 84109

• Phone: +1 801-438-1036
• Email: info@co-dx.com
• Website: www.co-dx.com

INTENDED USE

The Vector Smart™ Zika, dengue, and chikungunya RUO is a research use only multiplex test, based on real-time PCR (qPCR) technology, for the simultaneous qualitative detection of the Zika (ZIKV), dengue types 1-4 (DENV), and chikungunya (CHIKV) specific RNA in mosquitoes.

For research use only (RUO). Not for use in diagnostics procedures.

RUO COMPONENTS

See Table 1 for a list of RUO components.

Table 1
RUO Components List

Lid

Color

| Component| Symbol| Catalog

Number

| Description| Amount
---|---|---|---|---|---
__

Brown

| Vector Smart™ VZDC Master Mix| __

MM

| __

VZDC-MM- 001

| Proprietary blend of

CoPrimers™ and PCR reagents

| 1×500 µL (100

reactions)

__| Vector Smart™ VZDC Positive

Control

| __

PC

| __

VZDC-PC- 001

| Proprietary blend of target templates| 1×500 µL (100

reactions)

| No Template Control|

NC

| __

VZDC-NC-001

| DNase/RNase-free water| 1×500 µL (100

reactions)

__| Vector Smart™ VZDC Extraction

Control

| __

EC

| __

VZDC-EC-001

| Proprietary blend of the IPC template| 1×500 µL (100

reactions)

RUO Product Code is VZDC-R-001. Contact Sales at 801-438-1036 ext. 01 or at www.co- dx.com/contact/ to order.

VECTOR SMART™ VZDC STORAGE, HANDLING, AND DISPOSAL

• The Vector Smart™ VZDC RUO is shipped on dry ice. The components of the RUO should arrive frozen. If one or more of the components are not frozen upon receipt, or are compromised during shipment, contact your distributor for assistance. Upon receipt of kit, laboratory should follow internal procedures for quality control.
• All components should be stored below -16°C upon arrival to prevent degradation of reagents.
• Repeated thawing and freezing of components (more than four times) should be avoided, specifically the master mix, as this might affect the performance of the assay. The reagents should be frozen in multiple aliquots if they are to be used intermittently.
• Co-Diagnostics recommends storage between +2°C and +8°C should not exceed a period of 4 hours.
• If you work in an area prone to power outages, it is recommended to have a back-up generator for your freezer as well as a temperature data log system to ensure that the Vector Smart™ VZDC test RUO remains frozen at -16°C.
• Protect Master Mix from light.
• Expired products should not be used, as the integrity of the components cannot be guaranteed.
• The product is not a biological waste. See Safety Data Sheets (SDS) for hazard classification. Disposal should be in accordance with applicable regional, national, and local laws and regulations.

WARNINGS AND PRECAUTIONS

WARNING!
Read these Instructions for Use carefully before using the product. Before first use check the components for integrity and frozenness upon arrival.

Users should pay attention to the following:

• Use of this product should be limited to personnel instructed and trained in the techniques of real-time PCR.
• Samples should always be treated as infectious and/or biohazardous. Use standard precautions.
• Wear protective gloves, lab coat, and eye protection when handling samples. Always wear gloves when handling RUO components.
• Always use DNase/RNase-free disposable pipette tips with filters.
• Use segregated working areas for sample preparation, reaction setup, and amplification/detection activities. The workflow in the laboratory should proceed in a unidirectional workflow. To prevent contamination, change PPE between areas.
• Store and extract positive materials (specimen, controls, and amplicons) separately from other reagents. Dedicate supplies and equipment to separate working areas and do not move them from one area to another.
• Consult appropriate Safety data Sheets (SDS) for safety. The SDS for the Vector Smart™ VZDC test RUO is provided with the shipment. If not provided with shipment the SDS can be retrieved from Co-Diagnostics website at the link: http://co-dx.com/resources/safety-data-sheets/
• Do not open the reaction tubes/plates post amplification.
• Do not autoclave reaction tubes/plates after the PCR, since this will not degrade the amplified nucleic acid and will risk contaminating the laboratory area.
• Do not use components of the RUO that have passed expiration date.
• Discard sample and assay waste according to your local safety regulations.

BACKGROUND INFORMATION

Zika Virus (ZIKV)

• About: The Zika virus was first isolated in 1947 in monkeys found in the Zika Forest in Uganda. The first human cases were discovered in 1952 and since then outbreaks throughout Africa, the Americas, Asia, and the Pacific have been reported. Zika virus was not thought to be endemically transmitted in the Americas until the emergence in Brazil in 2015. There was a striking increase in reports of congenital microcephaly cases, which triggered a declaration of an international public health emergency (Araújo, et al., 2018). In this study Araújo et al. found direct correlation between microcephaly cases and Zika occurrences. Another study conducted in 2016 demonstrated that ZIKV infects and destroys human neuronal stem cells grown as neurospheres and brain organoids. These observations helped solidify the link between fetal ZIKV infection and the development of microcephaly (Relich & Loeffelholz, 2017). Due to these serious neurological sequelae found in 2018, the World Health Organization (WHO) issued the annual review of diseases where the priority for R&D investments for Zika has been raised (World Health Organization, 2018).
• The virus: is an enveloped, single-stranded (+) RNA virus part of the Flaviviridae family.
• Transmission: It is an arbovirus spread by the bite of infected mosquitos of the genus Aedes. Aedes mosquitoes usually bite during the day (early morning and late afternoon/evening). Zika virus is also transmitted from mother to fetus during pregnancy, through sexual contact, transfusion of blood and blood products, and organ transplant (Zika virus, 2018).

Dengue Virus (DENV)

• About: According to the World Health Organization Severe dengue was first recognized in the 1950s during the dengue epidemics in the Philippines and Thailand. Today, severe dengue affects most Asian and Latin American countries and has become a leading cause of hospitalization and death among children and adults in these regions (Dengue and severe dengue, 2019). There are 4 known serotypes of dengue (DENV 1-4). Immunity is acquired for long periods for each type, although the immunity to one type does not prevent the infection from the other types, what may cause in hyperendemic areas infection of dengue virus as frequent as up to four times per individual. The illness spectrum varies from asymptomatic, classic dengue fever, and severe or hemorrhagic dengue fever, which can be fatal (Wilder-Smith & Gubler, 2008). There is no specific treatment for dengue or severe dengue, but early detection and access to proper medical care lowers fatality rates below 1%. In many countries dengue fever is a reportable disease and must be registered with the responsible authority.
• The virus: is an enveloped, single-stranded (+) RNA virus part of the Flaviviridae family.
• Transmission: Like Zika virus, dengue is an arbovirus spread by the bite of infected mosquitos of the genus Aedes. Aedes mosquitos usually bite during the day (early morning and late afternoon/evening). Humans who are already infected with the virus can transmit the infection (for 4-5 days; maximum 12) via bite from uninfected Aedes mosquitoes after their first symptoms appear (Dengue and severe dengue, 2019).

Chikungunya virus (CHIKV)

• About: Chikungunya is a mosquito (mainly by Aedes albopictus) spread disease that leads to painful rheumatic symptoms. The lineages of chikungunya are divided into two branches: West African (WA) and East/Central/South African
  (ECSA). The West African strains are more related to small outbreaks locally in Africa. The strains from ECSA lineage are the ones that spread largely through the world (Silva & Dermody, 2017).

• The virus: is a small enveloped, single-stranded (+) RNA virus part of the Alphavirus genus of the family Togaviridae.

• Transmission: Like Zika and dengue virus, chikungunya is an arbovirus spread by the bite of infected mosquitos of the genus Aedes. Aedes mosquitos usually bite during the day (early morning and late afternoon/evening).

PRODUCT DESCRIPTION

The Vector Smart™ VZDC RUO is a research-use-only multiplex test, based on real-time polymerase chain reaction technology. It tests for the presence or absence of ribonucleic acid (RNA) of the Zika, Dengue types 1-4, and Chikungunya viruses. The Vector Smart™ VZDC test includes an internal control to identify possible qPCR inhibition, confirm the integrity of the reagents, and verify the quality of sample extraction. The Vector Smart™ VZDC test also includes a positive control which includes four synthetic nucleic acid molecules carrying sequences that are homologous to Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses and the mosquito gDNA positive control sequence are targeted by this multiplex assay. Positive controls represent a source of cross-contamination. Precautions should be taken to prevent and minimize the risk.
CoPrimers™ in the Vector Smart™ VZDC test include the following:

• CoPrimers™ that are targeting ZIKV are labeled with the FAM™ fluorophore
• CoPrimers™ that are targeting DENV are labeled with the CAL Fluor® Orange 560 fluorophore
• CoPrimers™ that are targeting CHIKV are labeled with the Quasar® 670 fluorophore
• CoPrimers™ that are targeting the Mosquito Enhancer of the Internal Positive Control (IPC) DNA are labeled with CAL Fluor® Red 610 fluorophore

MATERIALS AND DEVICES (REQUIRED BUT NOT PROVIDED)

• Appropriate 4-channel real-time PCR instrument, compatible with the fluorophores used in this test.
• Appropriate nucleic acid extraction system or kit, with associated equipment according to the manufacturer’s protocol.
• Vortex mixer
• Centrifuge with a rotor for 2 mL reaction tubes
• Pipettes (adjustable)
• Pipette tips with filters (disposable)
• Powder-free gloves (disposable)
• Ice
• Biosafety cabinet, ideally BSL-2 facility
• Copper-coated premium BBs (for extraction) or another sample homogenizer

WARNING!
All instruments should be properly installed, calibrated, and maintained according to the manufacturer’s instructions and recommendations. Do not use instruments with outdated calibration.

PROCEDURE

Mosquito Collection

Mosquitos are typically collected using commercially available mosquito traps, such as the CDC miniature light trap Model 512. The mosquitoes collected from a single collection site are often called a pool. The pool of mosquitoes are sexed and speciated based upon the region and the target(s) being tested for. After being sexed and speciated, the mosquitoes are either stored frozen or can go through the extraction process. After extraction, the mosquito extract can then be tested or stored frozen, preferably at -70°C for future testing.

Mosquito Preparation

The quality of the extraction of the RNA from the samples is essential for the performance of Vector Smart™ VZDC. The extraction protocol to be followed should be performed following the manufacturer’s instructions or an internally validated protocol. The suitability of the nucleic acid extraction procedure for use with Vector Smart™ VZDC must be validated by the user. Suggestions of extraction methods and systems include:

• QiAamp® Viral RNA Mini Kit (QIAGEN)
• MagMAX™ Viral RNA Isolation Kit (Applied Biosystems)
• MagMAX™ Viral Pathogen Nucleic Acid Isolation Kit (Applied Biosystems)
• Sbeadex Livestock Kit (LGC)

To prepare the mosquitoes before the extraction, place a pool of 10-50 mosquitoes in a 1.5 or 2.0 mL microcentrifuge tube, and add 10 µL per mosquito of (TE Buffer with 1% Trition X-100) to the tube (ex. 10 mosquitoes = 100 µL TE/Trition X mix added), and 1 copper coated premium BB (for 19 or less mosquitoes) or 2 BB’s (for 20 or more mosquitoes). Vortex the tube for 7 minutes, and centrifuge at 21,380 x g for 10 minutes. Remove the supernatant and continue with the extraction process.

WARNING!
An important step to ensure that the extraction process is working is to add 5 μL of Extraction Control, after the lysis step or when instructed by the extraction kit, into every sample pool being extracted. Due to the variability of mosquito populations, this will ensure that there is consistent amplification of the Mosquito Internal Positive Control (IPC).

For additional information and Technical Support regarding preparation please contact Technical Support 801-438-1036 ext. 02 or via email at support@co-dx.com .

WARNING!
If your sample preparation system is using washing buffers containing ethanol, make sure to eliminate any traces of ethanol prior to elution of the nucleic acid. Ethanol is a strong inhibitor of real-time PCR. The use of carrier RNA is crucial for extraction efficiency and stability of the extracted nucleic acid. Do not use buffer from other products besides the buffer in the sample extraction kit. Products like the RAMP grinding buffer is known as a PCR inhibitor and should not be used (Burkhalter, Horiuchi, Biggerstaff, Savage, & Nasci, 2014).

Set Up the Reagent
Perform the steps below to set up the reagent.

1. Clean all working surfaces with a fresh 10% bleach solution followed by a molecular-grade alcohol or another equivalent method of cleaning that disinfects and degrades nucleic acids.
2. Thaw all reagents and samples on ice, or a cold block, before starting the setup.
3. Vortex all Vector Smart™ VZDC DS MM, PC, nuclease-free water (used as a no template control or NC), and all sample tubes for 3 seconds.
4. Briefly spin the MM, PC, NC down before using to ensure reagents are properly mixed and to ensure removal of any condensation or residue from the lids.

Set Up the Reaction
Perform the steps below to set up the reaction.
Collect enough reaction wells for each of the following:

• One for each NC,
• One for each sample you want to test, and
• One (or more) for each PC

Note: The example below shows the number of wells needed for 5 known samples.

• PC 1
• NC 1
• Unknown samples 5
• Total wells required 7

Important:

• Pipette on ice, if possible.
• Perform PC pipetting and sample setup in a separate area, or at a separate time from the MM and NC.
• Change pipette tips between samples and change pipette tips after pipetting each component.
• Pipet the PC last, if possible, to avoid contamination events.

Pipet 5 μL of MM into each well collected.
Pipet 5 μL of the sample or 5 μL of NC control to the appropriate wells (in addition to the 5 μL of MM already in the well).

Note: Ensure to include at least one NC control in each run and that enough space remains for at least one PC.

• Pipet 5 μL of PC into the appropriate well.
• Seal the reaction plate with an optical adhesive film or seal each reaction tube with its appropriate lid.
• Place the plate or tubes into the real-time PCR instrument in the correct orientation and start the run.

PCR Instrument Setup

1. For basic information regarding setup and programming of the different real-time PCR instruments, please refer to the user manual of the respective instrument. For programming instructions questions regarding the use of other real-time PCR instruments please contact the Laboratory 801-438-1036 ext.03 or at www.co-dx.com/contact/.
2. If using a Co-Diagnostics Inc. CoDx Box, contact the Laboratory 801-438-1036 ext. 03 or at www.co-dx.com/contact/ for the template file for download. The template file comes pre-programmed with the settings described below. When not using a template, or using another device, use the settings outlined below to program the PCR instrument.
3. To achieve optimal performance from the test, it is important to make sure that the instrument is compatible with the conditions outlined in Table 2.

Table 2
Recommended Instrument Settings

__

Item

| __

Setting

---|---
__

Reaction Volume

| __

10 µL

__

Ramp Rate

| __

Default

__

Passive Reference

| __

None

Program PCR instrument with the cycling conditions in Table 3.

Table 3
Recommended Cycling Conditions

minutes

Initial Denaturation| Hold| 1| 95˚C| 2

minutes

__

Amplification

| __

Cycling

| __

50

| 95˚C| 3

seconds

55˚C| 32

seconds

• Ensure that PCR instrument being used is compatible with the fluorophores below. Some devices may not have options for the quencher. If needing help or have questions, contact Co-Diagnostics Inc. Technical Support at 801-438-1036 ext. 03 or at www.co-dx.com/contact/.
• Define the fluorescence detectors (dyes) as outlined in Table 4.

Table 4
Recommended Fluorescence Detector Definitions

__

Target

| __

Detector Name

| __

Reporter

| __

Quencher

---|---|---|---
__

ZIKV specific RNA

| __

ZIKV

| __

FAM™

| __

BHQ® – 1

__

DENV specific RNA

| __

DENV

| __

CAL Fluor® Orange 560

| __

BHQ® – 1

__

CHIKV specific RNA

| __

CHIKV

| __

Quasar® 670

| __

BHQ® – 2

__

Mosquito Internal Positive Control

| __

IPC

| __

CAL Fluor® Red 610

| __

BHQ® – 2

DATA ANALYSIS

For basic information regarding data analysis on specific real-time PCR instruments refer to the user manual of the respective instrument.

1. Validity of Test Runs
2. Valid Test Run
3. Check to see that both the positive and no template control passed.
4. The control conditions outlined in Table 5 must be met.

Table 5
Control Conditions to be Met

__

Control Type

| __

Control Name

| __

Purpose of Control

| __

ZIKV

| __

DENV

| __

CHIKV

| Internal Positive Control
---|---|---|---|---|---|---
__

__

__

__

VZDC Positive Control

| __

ZIKV (FAM™)

| __

__

__

Verifies the performance of the master mix

| __

__

__

__

| __

__

__

__

| __

__

__

__

| __

__

__

__

DENV (CF®560)
CHIKV (Q®670)
IPC (CF®610)
No Template Control| Master Mix + Water| Verifies the reagents are free of contamination| __

–

| __

–

| __

–

| __

–

If controls pass, interpret the sample results.

Invalid Test Run

1. If any of the controls fail, the run is invalid. Document the run and initiate troubleshooting.

Interpretation of Results
Once the controls have passed, the unknown samples can be interpreted based on three possible outcomes:

• Positive
• Negative
• Inconclusive

A Positive result will show an amplification curve or cycle threshold value for ZIKV, DENV, or CHIKV. The cut off value should be determined by in house validation testing. However, internal studies have shown rare primer-dimer formation or other non-specific amplification at 45 cycles. This fact can be attributed to the nature of the CoPrimers™ (Satterfield, 2014) (Poritz & Ririe, 2014). The amplification of the IPC shows that the extraction was successful. A Negative result will show no amplification for ZIKV, DENV, or CHIKV; The absence of a curve for VZDC indicates a negative result ONLY when the IPC marker is positive. An Inconclusive result will result if any of the controls fail. See troubleshooting. The interpretation of results can be translated to Table 6.

Table 6
Interpretation of Results Translation

__

__

Marker

| __

__

ZIKV

| __

__

DENV

| __

__

CHIKV

| Mosquito Internal Positive Control (NAM.18S)| __

Vector Smart™ Positive Control

| No Template Control (NC) Vector Smart™ Master Mix + Nuclease-Free

Water

| __

__

Result

---|---|---|---|---|---|---|---
__

Instrument Reading

| +| +| +| __

__

__

__

__

__

__

__

__

__

Pass

| VZDC +
–| –| –| VZDC –
__

+

| __

–

| __

–

| ZIKV +
DENV-
CHIKV-
__

–

| __

+

| __

–

| ZIKV-
DENV+
CHIKV –
__

–

| __

–

| __

+

| ZIKV-
DENV-
CHIKV+
__

+

| __

+

| __

–

| ZIKV+
DENV+
CHIKV-
__

–

| __

+

| __

+

| ZIKV-
DENV+
CHIKV+
__

+

| __

–

| __

+

| ZIKV+
DENV-
CHIKV+
__

__

__

Any Result

| __

Fail

| __

Pass

| Inconclusive: See Troubleshooting
__

__

Pass

| Fail| Pass|
Pass| Fail

The cut-off value will determine what results are to be considered positive or negative. It should be determined by the assay development.

TROUBLESHOOTING

Co-Diagnostics Inc. values customer feedback and we would like to be informed of any issues with the Vector Smart™ VZDC kit, even if the recommended steps for troubleshooting solve the issue. To give feedback please fill out the Customer Feedback Form by visiting www.co- dx.com/contact/feedback/

Stability
Real-time and accelerated shelf-life and in-use stability studies are currently under testing. Presently, the expiration date of this product has been established as 12 months. It is not recommended to use expired RUO reagents, doing so may lead to inaccurate results. Always use the most recent version of this document for updates as more stability information will be added when studies are completed.

User Errors
Good Laboratory Practices for Molecular Biology Diagnostics (Viana & Wallis, 2011) are necessary for the use of this product. This product is not intended to be used by untrained personnel. It is essential for the user to have some molecular biology experience and be familiar with proper pipetting technique to prevent errors, such as splashes, crossover contamination, and errors on volume selection. Pipette tips must be replaced after every pipetting. Gloves must be replaced often. Equipment, such as pipettes and real-time PCR instruments, should be calibrated when applicable. 90 minutes of online training for Good Laboratory Practices for Molecular Genetics Testing (Centers for Disease Control and Prevention, 2017) is available at the CDC website at the following link https://www.cdc.gov/labtraining/training-courses/good-lab- practices-molecular-genetics-testing.html

Invalid Results/Inconclusive Results

Vector Smart™ VZDC Positive Control not amplifying No amplification from the PC could be the result of one or multiple factors, such as:

• Pipetting errors (pipetting control into the wrong well, missing a well, pipetting inadequate amount of reagent),
• Incorrect placement of plates or tubes into the real-time PCR instrument,
• Vector Smart™ VZDC Master Mix or Vector Smart™ VZDC Positive Control degradation (result of reagents being at temperatures above -20°C for an extended period),
• Use of expired reagents,
• or the wrong reagents being used.

Without further evidence, it is best to disregard the results from the samples and re-test by re-amplification. If the positive control fails again, then an investigation should be conducted to identify possible causes for error, and the test must be reprocessed from extraction or not, depending on the investigation results and risks identified in the process. If failure of the positive control happens a third time after re-extraction and re- amplification, open a new Vector Smart™ VZDC Positive Control or Master Mix, and retest. If still failing, please contact Co-Diagnostics Inc. Technical Support at 801-438-1036 ext. 02 visiting www.co-dx.com/contact/.

NAM. 18s Mosquito Internal Positive Control (IPC) not amplifying in samples
No amplification from the IPC channel could be the result of one or multiple factors, such as:

• Not enough nuclear material in the sample,
• PCR inhibitors such as: ethanol and heparin,
• the extraction was performed incorrectly,
• or the extraction used is not compatible or has a step that eliminates a crucial element for extraction.
• Sample origin not compatible to the test designed application.
• or the extraction used is not compatible or has a step that eliminate the mosquito DNA (e.g., a DNase Digestion step.

Negative results, without IPC amplification, cannot be trusted and re-testing by re-amplification should be performed. If the IPC fails again, then samples should be re-extracted and re-amplified. If it fails after a third time an investigation should be conducted to identify possible causes for error. If the cause for the error is clear, the test can either be signed out as inconclusive due to either PCR inhibitors being present or not enough nuclear material being present. If the cause for error is unclear, contact Co- Diagnostics Inc. Technical Support by calling 801-438-1036 ext. 02 or by visiting www.co-dx.com/contact/.

No Template Control showing the amplification

• Amplification of VZDC in a No Template Control indicates contamination in one or more of the reagents, incorrect placement of plate or tube into the real-time PCR instrument, or pipetting errors. None of the results can be trusted and re-testing by re-amplification should be performed. If the NC fails again, then an investigation should be conducted to identify possible causes for error, and the test must be reprocessed from extraction or not, depending on the investigation results and risks identified in the process. If failure of the NC, after re-extraction and re-amplification, happens a third time, open a new nuclease-free water and retest. If still failing, please contact Co-Diagnostics Inc. Technical Support by calling 801-438-1036 ext. 02 or by visiting www.co-dx.com/contact/.

LIMITATIONS

• This product is intended for research use only. Not intended for use in clinical diagnostics for its performance for diagnostic applications has not to been established.
• Strict compliance with this document is required for optimal results. Please, always use the most recent version of this document. This can be downloaded for free at: www.co-dx.com/resources/instructions-for-use/ or by visiting www.co-dx.com/contact/.
• Use of this product is to be limited to trained and instructed personnel in real-time PCR techniques.
• Good laboratory practices are essential for the proper performance of this assay. It is also recommended that upon receipt of reagents a test run be performed to check the integrity, and performance of the reagents prior to testing on samples.
• Appropriate collection, transport, storage, and processing procedures of samples are required for optimal results.
• Do not use the Vector Smart™ VZDC RUO components directly on the specimens collected. Perform an appropriate nucleic acid extraction prior to using this assay.
• The presence of PCR inhibitors may cause false negatives or invalid results.
• Potential mutations of the target regions of the ZIKV, DENV, and CHIKV genome covered by this test RUO may result in failure to detect the presence of the pathogens.

QUALITY CONTROL

In accordance with Co-Diagnostics Inc.’s ISO 13485-certified Quality Management System, each lot of Vector Smart™ VZDC RUO is tested against predetermined specifications to ensure consistent product quality.

TECHNICAL ASSISTANCE

For technical assistance, please contact our Technical Support:

• Website: http://co-dx.com/contact/
• Email: info@co-dx.com
• Phone: 801-438-1036 ext. 02

REFERENCES

Araújo, T. V., Ximenes, R. A., Miranda-Filho, D. d., Souza, W. V., Montarroyos, U. R., Melo, A. P., . . . Rodrigues, L. C. (2018, March 1). Association between microcephaly, Zika virus infection, and. The Lancet Infectious Diseases, 328–336. doi:https://doi.org/10.1016/S1473-3099(17)30727-2 Burkhalter, K. L., Horiuchi, K., Biggerstaff, B. J., Savage, H. M., & Nasci, R. S. (2014). Evaluation of a Rapid Analyte Measurement Platform and Real-Time Reverse-Transcriptase Polymerase Chain Reaction Assay West Nile Virus Detection System in Mosquito Pools. Journal of the American mosquito Control Association, 30(1), 21-30. Centers for Disease Control and Prevention. (2017, Oct 27). CDC Laboratory Training: Good Laboratory Practices for Molecular Genetics Testing. Retrieved Mar 5, 2019, from CDC: https://www.cdc.gov/labtraining/training-courses/good- lab-practices-molecular-genetics-testing.html Dengue and severe dengue. (2019, April 15). Retrieved from World Health Organization: https://www.who.int/news-room/fact- sheets/detail/dengue-and-severe-dengue Poritz, M. A., & Ririe, K. M. (2014). Getting Things Backwards to Prevent Primer Dimers. The Journal of Molecular Diagnostics, 16(2), 159-162. doi:10.1016/j.jmoldx.2014.01.001 Relich, R. F., & Loeffelholz, M. (2017). Zika Virus. Clinics in Laboratory Medicine, 37(2), 253-267. doi:10.1016/j.cll.2017.01.002 Satterfield, B. (2014, Mar). Cooperative primers: 2.5 million-fold improvement in the reduction of nonspecific amplification. The Journal of Molecular Diagnostics, 16(2), 163-173. doi:10.1016/j.jmoldx.2013.10.004 Silva, L. A., & Dermody, T. S. (2017, Mar 1). Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies. The Journal of Clinical Investigation, 127(3), 737-749. doi:10.1172/JCI84417 Viana, R. V., & Wallis, C. L. (2011). Good Clinical Laboratory Practices (GLCP) for Molecular Based Tests Used in Diagnostic Laboratories. In D. I. Akyar, Wide Spectra of Quality Control (pp. 29-52). InTech. Retrieved from http://www.intechopen.com/books /wide-spectra-of-quality-control/goodclinical-laboratory-practice-gclp-for- molecular-based-tests-used-in-diagnostic-laboratories
Wilder-Smith, A., & Gubler, D. J. (2008, Nov 1). Geographic Expansion of Dengue: The Impact of international Travel. Medical Clinics of North America, 92(6), 1377-1390. World Health Organization. (2016, March 23). Laboratory testing for Zika virus infection. Retrieved September 15, 2018, from World Health Organization: http://apps.who.int/iris/bitstream/handle/10665/204671/WHO_ZIKV_LAB_16.1_eng.pdf?sequence=1

World Health Organization. (2018). 2018 Annual review of disease prioritized under the Research and Development Blueprint. Retrieved September 15, 2018, from http://www.who.int/emergencies/diseases/2018prioritization- report.pdf?ua=1
Zika virus. (2018, July 20). Retrieved from World Health Organization: https://www.who.int/en/news-room/fact-sheets/detail/zika-virus

LEGEND OF PACKAGE SYMBOLS

See Table 7 for a legend of the package symbols.

Table 7
Legend of Package Symbols

2401 S. Foothill Dr. Ste. D., Salt Lake City, UT 84109, USA

• 801.438.1036
• www.co-dx.com

References

• Contact Us | Co-Dx - Co-Diagnostics, Inc.
• Safety Data Sheets - Co-Diagnostics, Inc.
• The Industry Leading Domain Broker - MediaOptions
• Molecular Tests | Leading-Edge PCR Technology | Co-Dx - Co-Diagnostics, Inc.
• Contact Us | Co-Dx - Co-Diagnostics, Inc.
• Zika virus
• Dengue and severe dengue

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