new york state department of health - wadsworth

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NYS DOH LEB-618, revision 2; 9/10/2020

New York State Department of Health - Wadsworth Center Laboratory of Environmental Biology

NYS ELAP Laboratory ID 10765

Division of Environmental Health Sciences Albany, New York

NYS DOH LEB-618

Set-Up and Analysis of qPCR Assays for Mold Identification in Medical Marijuana Products

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Contents

1.0. Scope and Application ..................................................................................... 3

2.0. Summary of the Method .................................................................................. 3

3.0. Definitions ...................................................................................................... 3

4.0. Health and Safety Warnings............................................................................ 4

5.0. Shipping Conditions, Receiving, Preservation and Storage.............................. 4

6.0. Interferences ................................................................................................... 5

7.0. Apparatus and Materials ................................................................................. 5

8.0. Quality Control/Assurance .............................................................................. 6

9.0. Procedure........................................................................................................ 7

10.0. Data Acquisition, Reduction, Analysis, Calculations, Acceptance Criteria and

Documentation .......................................................................................................... 10

11.0. Method Performance..................................................................................... 11

12.0. Waste Management/Pollution Prevention...................................................... 11

13.0. References..................................................................................................... 11

14.0. Appendices .................................................................................................... 12

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1.0. Scope and Application

1.1. This method, NYS DOH LEB-618, Set-Up and Analysis of qPCR Assays for Mold Identification in Medical Marijuana Products (ELAP METHOD ID 9977) describes how to set up and analyze real time, quantitative PCR (qPCR) assays in a 96-well plate format using TaqMan® enzyme technologies in medical marijuana samples as

required in Title 10 (Health), Subpart 55-2.15 and Chapter XIII, Section 1004.14 of the

official Compilation of Codes, Rules, and Regulations, of the State of New York. 1.2. This procedure is used for the identification of molds belonging to the

Mucor/Rhizopus group, Penicillium/Paecilomyces spp. or Aspergillus spp. 1.3. It is applicable to DNA prepared according to NYS DOH LEB-609 from mold

colonies isolated from Medical Marijuana products according to NYS DOH LEB-

605. 1.4. Protocols for the identification of these organisms in samples of medical marijuana

products can be found in the NYS DOH LEB-600 series. See Medical Marijuana

Microbial Testing Plan flowcharts

2.0. Summary of the Method

2.1. Stock and working solutions of primers and probes are made and stored appropriately.

2.2. A master mix is prepared and aliquoted into separate wells of a 96-well plate. 2.3. Sample DNA, prepared in NYS DOH LEB-609, is added to the master mix and

subjected to qPCR on a Applied Biosystems Fast 7500 thermocycler. 3.0. Definitions

3.1. qPCR stands for real-time, quantitative, polymerase chain reaction.

3.2. Target sequence refers to a segment of a gene which will be amplified using qPCR. 3.3. A forward primer is a short DNA sequence which is complementary to the 5’ end

of the target sequence and is used to initiate DNA replication using qPCR. 3.4. A reverse primer is a short DNA sequence which is complementary to the 3’ end of

the target sequence and is used to initiate DNA replication using qPCR. 3.5. A probe is a short DNA sequence which is complementary to the target sequence 5

to 10 base pairs downstream of the forward primer. The probe is labeled with a fluorescent dye and quencher dye to allow for detection of amplification in a

TaqMan®-based qPCR assay. 3.6. BSA stands for bovine serum albumin, fraction V powder. 3.7. TaqMan® Environmental Master Mix or PerfeCTa® qPCR ToughMix® are

commercially prepared mixes of DNA Polymerase, UP (Ultra Pure), dNTPs,

AmpErase® UNG, optimized buffer, and the passive internal reference dye ROX™. 3.8. A master mix (MM) consists of working concentrations of primers and probes,

BSA, water, and either TaqMan® Environmental Master Mix or PerfeCTa® qPCR ToughMix®.

3.9. A positive control for qPCR is a reaction containing DNA that will be amplified using the primer/probe set.

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3.10. A negative control for qPCR is a reaction containing DNA that will not be

amplified using the primer/probe set. 3.11. NTC stands for no template control which is a qPCR reaction containing water

instead of DNA. 3.12. A process control sample is a sample containing only extraction buffer which is

extracted and analyzed in conjunction with the unknown samples (see NYS DOH LEB-609).

3.13. RO stands for an organization that is registered to manufacture and dispense medical marijuana in New York State.

4.0. Health and Safety Warnings 4.1. Microbiological analyses involve the culturing of potentially pathogenic organisms.

4.1.1. All microbiologically contaminated materials, including media, shall be

autoclaved after use. 4.1.2. Contaminated glassware and plastic ware shall be decontaminated prior to

washing. 4.1.3. Laboratory equipment and benches shall be disinfected using either

Envirocide®, 10% bleach, or a minimum concentration of 70% ethanol before and after use.

4.1.4. Mouth pipetting is prohibited. 4.1.5. All accidents, particularly those which may result in infection, shall be

reported according to laboratory-specific policies and procedures. 5.0. Shipping Conditions, Receiving, Preservation and Storage

5.1. Sample shipping conditions

5.1.1. The medical marijuana products from the Registered Organizations (ROs) are shipped as per manufacturer’s specifications and must adhere to all regulatory requirements.

5.2. Sample Receipt

5.2.1. Medical marijuana products from the RO are received, verified and documented ensuring that method, regulatory and Accreditation Body requirements are met.

5.3. Method holding times

5.3.1. This procedure is initiated upon completion DNA extraction in the Mold Identification SOP (see NYS DOH LEB-609).

5.4. Preservation. 5.4.1. Sabouraud Dextrose Agar sample plates containing mold colonies are

stored refrigerated until it has been determined that they are not needed for additional microbiological evaluation.

5.4.2. 96-well qPCR plates containing master mixes and sample DNA can be stored refrigerated for several hours prior to running the plates.

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5.5. Storage

5.5.1. If storage is required prior to analysis, isolates or archived plates are stored refrigerated until it has been determined that they are not needed for additional microbiological evaluation.

6.0. Interferences

6.1. Some components of medical marijuana products, e.g., ethanol, may inhibit the growth of microorganisms.

7.0. Apparatus and Materials

7.1. Equipment

7.1.1. Automatic pipetters and sterile aerosol-resistant micropipette tips 7.1.2. 1.7mL microcentrifuge tubes, sterile 7.1.3. 96-well plates specific to the ABI 7500Fast instrument – Applied

Biosystems, Cat. No. 4346906

7.1.4. 96-well plate cooler, stored at -20oC; Krackeler, Cat. No. 38-022510525, or equivalent

7.1.5. Optical plate covers – Applied Biosystems, Cat. No. 4311971 7.1.6. Balance capable of accuracy to 0.01g

7.1.7. Rotamix rotating mixer – Appropriate Technical Resources, Laurel MD, cat no. RKVSD, or equivalent.

7.1.8. Vortex 7.1.9. Eppendorf 5430 Microplate Centrifuge with 96-well plate adapter,

Krackeler Cat. No. 38-022620568, or equivalent. 7.1.10. Syringe filters, 0.22 micron, Fisher Scientific, Cat. No. 09-754-13, or

equivalent. 7.1.11. Syringe with Luer-Lok tips, 10mL Fisher Scientific, Cat. No. 14-823-16E,

or equivalent. 7.1.12. NIST traceable weights 7.1.13. Balance 7.1.14. AirClean PCR Work Station, AirClean, Cat. No. AC632LFUVC, or

equivalent equipped with a dedicated set of micropipettors and microcentrifuge.

7.2. Reagents and Chemicals 7.2.1. TaqMan® Environmental Master Mix, Applied Biosystems Cat. no.

4398021 or PerfeCTa® qPCR ToughMix® UNG, Low ROX, Quanta Biosciences Cat. no 95140-012.

7.2.2. Bovine serum albumin (BSA), fraction V powder, Sigma Cat. no. B-4287 or equivalent.

7.2.3. Forward primer (sequences vary among targets, see LEB-AP-618D) 7.2.4. Reverse primer (sequences vary among targets, see LEB-AP-618D)

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7.2.5. Probe (sequences vary among targets, see LEB-AP-618D)

7.2.6. PCR grade water, OmniPur water from VWR EM-9610 or equivalent. Water must be DNase/RNase free.

7.3. Forms

7.3.1. Mold Identification Results Sheet (e.g., LEB-RS-609B)

7.3.2. Mold Identification qPCR Targets and Positive Controls Appendix (see LEB-AP-618D)

7.3.3. Mold qPCR Analyses Bench Sheet (e.g., LEB-RS-618A) 7.3.4. Setting Up a 7500 Fast Template to Detect Molds Appendix (see LEB-

AP-618A) 7.3.5. How to Start and Analyze qPCR Analyses to Detect Molds Appendix (see

LEB-AP-618B) 7.3.6. Reconstituting Oligonucleotides Appendix (see LEB-AP-618C)

8.0. Quality Control/Assurance 8.1. Method Detection Limits

8.1.1. Method Detection Limits are product-specific.

8.2. Calibration and Standardization

8.2.1. Temperatures of the cold room and refrigerators are observed and recorded twice daily separated by at least 4 hours.

8.2.1.1. If the cold room or refrigerator does not stay within 1.0-8.0°C, laboratory-specific corrective actions are followed.

8.2.1.2. The optimum temperature range for a cold room or refrigerator is 1.0-4.0°C

8.2.1.3. If the cold room or refrigerator was in a defrost cycle at the time that the temperature was recorded, and the temperature does not reach 8.0°C, re-testing of media is not required.

8.2.1.4. Supplies may be re-tested for quality, depending on the number

of degrees and the amount of time that the cold room or refrigerator temperature was out of compliance, at the discretion of the laboratory.

8.2.2. Temperature of the freezer is observed and recorded twice daily separated

by at least 4 hours. 8.2.2.1. If the temperature on the freezer exceeds -15.0°C, laboratory-

specific corrective actions are followed. 8.2.3. Max/min temperatures are recorded when twice-daily temperature

measurements are not possible, such as on holidays and weekends. 8.2.4. Thermometers must be calibrated as prescribed by the Accreditation Body

and in accordance with relevant regulations and standards. 8.2.5. The volumetric accuracy of automatic pipettes and serological pipettes is

verified as prescribed by the Accreditation Body in accordance with relevant regulations and standards.

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8.2.6. The intensity and efficacy of the UV light in the AirClean PCR hood is

determined in accordance with relevant regulations and standards. 8.2.7. The balance is calibrated with NIST-traceable weights prior to use.

8.3. Quality Control

8.3.1. A concentration of 200ng/mL of Salmon Testes DNA (Sketa) is added to

each DNA extract (see NYS DOH LEB-609 section 9.2.3) as an RT-PCR internal control to detect amplification inhibition. If the cycle threshold (CT) value for a sample is greater than 25, and the Sketa CT in a sample is 2 cycles greater than the extraction buffer control, the amplification of the

sample DNA is considered inhibited. Samples with amplification inhibition are diluted and re-analyzed.

8.3.2. Each qPCR assay contains positive control DNA, negative control DNA, extraction control DNA, and a no template control.

8.4. Corrective/Preventive Actions

8.4.1. The laboratory will initiate non-conformances and/or corrective/preventive actions in accordance with laboratory-specific procedures and as prescribed by the Accreditation Body and in accordance with relevant

regulations and standards.

9.0. Procedure

9.1. Preparation of Solutions

9.1.1. PCR Grade Water

9.1.1.1. Dispensing PCR grade water is performed in a PCR workstation to ensure sterility.

9.1.1.2. PCR grade water is aseptically aliquoted into sterile 1.7mL

microcentrifuge tubes and stored long term at -20°C. 9.1.2. Bovine Serum Albumen (BSA)

9.1.2.1. All stock solutions of BSA are 2mg/mL concentration

9.1.2.2. Place an empty, sterile 15mL conical tube on the balance and

tare the balance. 9.1.2.3. Using a flame-sterilized spatula, add 20mg of BSA to the 15mL

conical tube. 9.1.2.4. Add enough PCR grade water to make a final volume 10mL.

9.1.2.5. Place the tube on a rotating mixer, and rotate for 2 hours, at room temperature, or until the BSA is completely dissolve into solution.

9.1.2.6. Once dissolved, filter-sterilize the BSA solution using a 0.22μm

syringe filter and 10mL syringe, and aliquot into 1.7mL tubes to be stored long term at -20°C.

9.1.2.6.1. Dispensing BSA is performed in a PCR workstation to ensure sterility.

9.1.3. Primer Stock Solutions – 1mM

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9.1.3.1. Preparation of primer stock solutions is performed in a PCR

workstation to ensure sterility. 9.1.3.2. All stock solutions of primers are prepared at a 1mM final

concentration. 9.1.3.2.1. Calculate how much PCR-grade water is needed to

create a 1mM concentration of primer by using an online calculator provided by many primer manufacturers. Use LEB-AP-618C as a guide

9.1.3.2.2. For example, go to the IDT website and use the

reconstitution calculator to determine the amount of PCR-grade water to add.

9.1.3.2.2.1. www. idtdna.com/Calc/resuspension/ 9.1.3.2.3. Record the amount of water to be added on the

Oligonucleotide Specification Sheet along with the date of resuspension and analyst initials.

9.1.3.2.4. Centrifuge the tube briefly. 9.1.3.2.5. Reconstitute with the appropriate amount of nuclease-

free PCR-grade water as determined above. 9.1.3.2.6. Invert the tube several times to mix gently and

centrifuge briefly. 9.1.3.2.7. Label the tube with the final concentration, the date of

reconstitution, and analyst initials. 9.1.3.2.8. Store stock solutions at -20°C indefinitely.

9.1.4. Primer/Probe Working Solutions

9.1.4.1. Preparation of working primer/probe solutions is performed in a

PCR workstation to ensure sterility. 9.1.4.2. Working primer/probe solutions have final concentrations of

1µM for each primer and 80nM for the probe. 9.1.4.2.1. Remove the stock solutions for the primers (prepared

according to section 9.1.3) and probes (from the manufacturer, provided at a prepared concentration of 100μM) from the freezer and thaw.

9.1.4.2.2. Flick each to mix gently, and briefly spin tubes down.

9.1.4.3. Label a sterile 1.7mL microcentrifuge tube with the target name, “Working”, and the date of preparation.

9.1.4.4. To the sterile 1.7mL microcentrifuge tube, add 686μL of DNase/RNase-free sterile water.

9.1.4.5. To the labeled tube add: 9.1.4.5.1. 5μL of the 1mM forward primer stock solution. 9.1.4.5.2. 5μL of the 1mM reverse primer stock solution. 9.1.4.5.3. 4μL of the 100µM probe stock solution.

9.1.4.6. Flick and invert the tubes several times to mix and centrifuge briefly.

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9.1.4.6.1. DO NOT vortex to mix.

9.1.4.7. Store the working stock solutions at 2-8°C until used.

9.2. Preparation of Master Mixes

9.2.1. Preparation of master mixes is performed in a PCR workstation to ensure sterility.

9.2.2. Master mixes are prepared immediately prior to plate set-up.

9.2.2.1. Thaw an aliquot of a PCR grade water (prepared according to section 9.1.1).

9.2.2.2. Thaw an aliquot of a 2mg/mL concentration of BSA (prepared according to section 9.1.2).

9.2.2.3. Label a sterile 1.7mL microcentrifuge with the target name and the designation “MM” for master mix.

9.2.2.4. Create a master mix for each sample, positive control, negative control, extraction control, no template control, and 2 extras to

account for lost volume during pipetting. 9.2.2.5. Each individual reaction includes:

9.2.2.5.1. 1.5μL of sterile PCR grade water (prepared in section 9.1.1)

9.2.2.5.2. 2.5μL of BSA (prepared in section 9.1.2.) 9.2.2.5.3. 3.5μL of the target-specific probe/primer working

solution (prepared in section 9.1.4.) 9.2.2.5.4. 12.5μL of either TaqMan® Environmental Master Mix

or Quanta PerfeCTa® qPCR ToughMix® UNG, Low ROX.

9.2.2.6. Invert the tubes several times to mix, and centrifuge briefly. 9.2.2.7. Place tube on ice until used.

9.2.2.7.1. Master mixes are made immediately prior to plate set up and should not be kept on ice for more than 1 hour.

9.2.3. Return the remaining target-specific probe/primer working solutions and Environmental Master Mix or ToughMix to the refrigerator.

9.3. Setting up a qPCR Plate for Sample Analyses

9.3.1. Setting up a qPCR plate for sample analyses occurs in a physically different location than the preparation of master mixes to ensure there isn’t

any cross contamination of sterile supplies. 9.3.2. Using the mock 96-well plate on the Mold qPCR Analyses Bench Sheet

(e.g., LEB-RS-618A), record the location of each sample and assay target in the 96-well plate.

9.3.3. Place a 96-well PCR plate in a 96-well plate ice block from the freezer, being careful not to touch the top of the plate or contaminate the wells.

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9.3.4. Using an automatic micropipettor and sterile tip and the mock 96-well

plate (see LEB-RS-618A as a guide), add 20μL of the appropriate master mix (prepared in section 9.2.) to the wells indicated on the bench sheet.

9.3.5. Using an automatic micropipettor and sterile tip, and the mock 96-well plate (see LEB-RS-618A as a guide), add 5μL of DNA from each sample

and positive control to each well indicated on the bench sheet. 9.3.6. Using an automatic micropipettor and sterile tip, and the mock 96-well

plate (see LEB-RS-618A as a guide), add 5μL of nuclease free water to the NTC well indicated on the bench sheet.

9.3.7. Once the plate set up is complete, cover the plate with a plate cover and press down to seal the plate.

9.3.8. Centrifuge the plate briefly by holding down the “short” button and allowing the centrifuge speed to reach at least 400rpm and release the

button. Do not spin the plate over 1,000rpm. 9.3.9. Place in the 7500Fast plate tray. 9.3.10. Turn on the 7500Fast thermocycler, and use LEB-AP-618A and LEB-AP-

618B as guides to run the qPCR assay.

9.4. Analysis of the qPCR Reactions

9.4.1. Use LEB-AP-618B as a guide for setting the threshold to 0.2. 9.4.2. Using LEB-AP-618A as a guide, record any CT value results for each

sample, positive control, negative control, and NTC on the Mold qPCR Analyses Bench Sheet (e.g., LEB-RS-618A). 9.4.2.1. Samples with CT values at 25 or lower are recorded positive on

the Mold Identification by qPCR Results Sheet (e.g., LEB-RS-

609B). 9.4.2.2. Samples with CT values of above 25 must be analyzed for DNA

amplification inhibition. 9.4.2.2.1. If a sample has a Sketa CT value at least 2 cycles higher

than the Sketa CT of the extraction buffer, the sample DNA amplification is considered inhibited.

9.4.2.2.2. Any samples with inhibition are diluted 1:5 in extraction buffer and re-analyzed.

9.4.2.3. Samples with CT values above 25 and do not have amplification inhibition are recorded as negative on the Mold Identification by qPCR Results Sheet (e.g., LEB-RS-609B).

9.4.2.4. Positive controls should have CT values less than 25.

9.4.2.5. Negative controls should be non-detects, or have a CT greater than 35.

10.0. Data Acquisition, Reduction, Analysis, Calculations, Acceptance Criteria and

Documentation

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10.1. Record sample ID, DNA extraction date, CT values, Sketa inhibition, data file

name, extraction tube lot, AE buffer lot, master mix type, master mix lot and expiration date, run date, and analyst initials on the Mold qPCR Analyses Bench Sheet (e.g., LEB-RS-618A).

11.0. Method Performance

11.1. Demonstration of Capability 11.1.1. Prior to acceptance and use of this method for data reporting, a

satisfactory initial demonstration of capability (DOC) is required. Thereafter, an ongoing DOC is to be performed annually.

11.1.2. An initial DOC shall be made prior to using any method, and at any time there is a change in instrument type, personnel or method or any time that a method has not been performed by the laboratory or analyst in a twelve (12) month period.

11.1.3. All DOCs shall be documented, and all data applicable to the demonstration shall be retained and readily available at the laboratory.

11.1.4. Consult relevant standards, regulations and Accreditation Body requirements for additional information on performing DOCs for

microbial contaminants. 11.2. Laboratory Detection Limits

11.2.1. See section 8.1.

12.0. Waste Management/Pollution Prevention

12.1. None.

13.0. References

13.1. EPA method A (EPA 821-R-10-004, April 2010). 13.2. TaqMan® Environmental Master Mix Product Bulletin. Applied Biosystems.

Publication 116PB11-03. 13.3. TaqMan® Environmental Master Mix Protocol. Applied Biosystems. 4448845

Rev. B 06/2010. 13.4. PerfeCTa® qPCR ToughMix™, Low Rox™ Protocol. Quanta Biosciences. 95114

Rev 110801. 13.5. NYS DOH LEB-609, Mold Plate Counts and Identification for Medical Marijuana

Testing

13.6. Title 10 (Health), Subpart 55-2.15 and Chapter XIII, Section 1004.14 of the official Compilation of Codes, Rules, and Regulations, of the State of New York

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14.0. Appendices

Appendix A – Mold Bench Sheets (LEB-RS-618A)

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Appendix A – Mold Bench Sheets (LEB-RS-618A) (con’t)

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Setting Up a 7500 Fast Template to Detect Molds (LEB-AP-618A)

Screenshot 1 (Step 1)

Screenshot 2 (Steps 2-4)

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Setting Up a 7500 Fast Template to Detect Molds (LEB-AP-618A) (con’t)


Screenshot 4 (Steps 6 and 7)

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Screenshot 6

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Screenshot 9


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Screenshot 12



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Screenshot 14


Screenshot 16

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Screenshot 19 (Steps 20-22)

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Start and Analyze RT-PCR Analyses to Detect Molds (LEB-AP-618B)

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Start and Analyze RT-PCR Analyses to Detect Molds (LEB-AP-618B) (con’t)

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Reconstituting Oligonucleotides (LEB-AP-618C)

1. Example of a Oligonucleotide Specification Sheet

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Reconstituting Oligonucleotides (LEB-AP-618C) (con’t)

2. Entering Values into the IDT Reconstitution Calculator Application

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3.0 Results from the IDT Reconstitution Calculator Application

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3.0 Results from the IDT Reconstitution Calculator Application (con’t)

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4. Recording Results on the Oligonucleotide Specification Sheet

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Mold Identification RT-PCR Targets and Controls Appendix (LEB-AP-618D)

Target Name: Pan-Asp1 (Aspergillus/Penicillium general) Forward Primer: 5’-gTggAgTgATTTgTCTgCTTAATTg-3’ Reverse Primer: 5’-TCTAAgggCATCACAgACCTgTT-3’

Probe: 5’-6FAM-CggCCCTTAAATAgCCCggTCCg-QSY-3’ Positive Control: A. brasiliensis ATCC 16404 and P. chrysogenum ATCC 11709 Negative Control:M. hiemalis ATCC 28932

Target Name: PenAsp1mgb2 (Aspergillus/Penicillium general) Forward Primer: 5’-CggAAggATCATTACTgAgTg-3’ Reverse Primer: 5’-gCCCgCCgAAgCAAC-3’

Probe: 5’-6FAM-CCAACCTCCCACCCgTg-MGBNFQ-3’ Positive Control: A. brasiliensis ATCC 16404 and P. chrysogenum ATCC 11709 Negative Control:M. hiemalis ATCC 28932

Target Name: Muc12 (Mucor/Rhizopus) Forward Primer: 5’-CACCgCCCgTCgCTAC-3’ Reverse Primer: 5’-CCTAgTTTgCCATAgTTCTCAgCAg-3’

Probe: 5’-6FAM-CCgATTgAATggTTATAgTgAgCATATgggATC-TAMRA-3’ Positive Control: M. hiemalis ATCC 28932 Negative Control:A. brasiliensis ATCC 16404 or P. chrysogenum ATCC 11709

Target Name: Aflav1 (A. flavus) Forward Primer: 5’-CgAgTgTAgggTTCCTAgCgA-3’ Reverse Primer: 5’-CCggCGgCCATgAAT-3’

Probe: 5'-FAM-TCCCACCCgTgTTTACTgTACCTTAgTTgCT-TAMRA-3' Positive Control: A. flavus ATCC 16883 Negative Control:M. hiemalis ATCC 28932 or P. chrysogenum ATCC 11709

Target Name: Afum1 (A. fumigatus) Forward Primer: 5'-gCCCgCCgTTTCgAC-3' Reverse Primer: 5'-CCgTTgTTgAAAgTTTTAACTgATTAC-3'

Probe: 5'-FAM-CCCgCCgAAgACCCCAACATg-TAMRA-3' Positive Control: A. fumigatus ATCC 34506 Negative Control:M. hiemalis ATCC 28932 or P. chrysogenum ATCC 11709

Target Name: Anigr1 (A. niger) Forward Primer: 5'-gCCggAgACCCCAACAC-3' Reverse Primer: 5'-TgTTgAAAgTTTTAACTgATTgCATT-3'

Probe: 5'-FAM-AATCAACTCAgACTgCACgCTTTCAgACAg-TAMRA-3' Positive Control: A. niger ATCC 16888 Negative Control:M. hiemalis ATCC 28932 or P. chrysogenum ATCC 11709

Mold Identification RT-PCR Targets and Positive Controls

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Appendix (LEB-AP-618D, con’t)

Target Name: Aterr1 (A. terreus) Forward Primer: 5'-ATCATTACCgAgTgCgTgTCTTTA-3' Reverse Primer: 5'-CCCgCCgAAgCAACAAg-3'

Probe: 5'-FAM-CCCAACCTCCCACCCgTgACTATTg-TAMRA-3' Positive Control: A. terreus ATCC 1012 Negative Control:M. hiemalis ATCC 28932 or P. chrysogenum ATCC 11709

Target Name: Sketa3 (Internal control) Forward Primer: 5’-ggTTTgTTgAggATgAgCTTCTg-3’ Reverse Primer: 5’-CgAAgCgAAAgggAAAggA-3’

Probe: 5’-FAM-CTACTTCCgTTCCCCCgTgCgC-TAMRA-3' Positive Control: Extraction buffer Negative Control:A. brasiliensis ATCC 16404 or M. hiemalis ATCC 28932 or P. chrysogenum

ATCC 11709

References for LEB-AP-618D:

1. Walsh, T.J., et. al. 2011. Molecular detection and species-specific identification of medically important Aspergillus species by real-time PCR in experimental invasive pulmonary aspergillosis. J. Clin. Micro. 49(12): 4150-4157.

2. Environmental Protection Agency (EPA). 2015. Microbiological and Chemical Exposure Assessment, EPA Technology for Mold Identification and Enumeration. Accessed [2015], at URL

[http://www.epa.gov/microbes/moldtech.htm.

new york state department of health - wadsworth

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