Animal & Veterinary

Development of a Multiplex Real-Time PCR Assay for the Detection of Ruminant DNA in Raw Materials used for Monitoring Crude Heparin for Quality

Draft

Sharla M. Peters1, Yolanda L. Jones1, Frank Perrella2, Tai Ha3, and Haile F. Yancy1

1U. S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research, 8401 Muirkirk Road, Laurel, Maryland 20708, USA. 2U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Compliance, Silver Spring, MD 20993, USA. 3Nebraska Department of Agriculture, 301 Centennial Mall South, Lincoln, Nebraska 68508, USA.

Abstract

A real-time PCR assay was developed for the determination of ruminant material in porcine-derived crude heparin products. The assay consists of a bead format with lyophilized primers and probe sets that identify ruminant (bovine, ovine, caprine) and porcine material and also contain an internal amplification control. The assay was verified by two analysts: the first located at the FDA and the second at an independent State laboratory. Performance of the assay was evaluated against stringent acceptance criteria developed by the U.S. FDA’s Center for Veterinary Medicine’s Office of Research. The heparin Multiplex Real-Time Assay (hMRTA) for the detection of ruminant DNA in porcine crude heparin passed the stringent acceptance criteria for specificity, sensitivity, and selectivity. The assay met sensitivity and reproducibility requirements previously established by the multiplex real-time PCR assay (MRTA) for detection of ruminant animal material in feed. The hMRTA, when used in porcine crude heparin, exhibited 98% sensitivity, 98% true positives, 2% false negatives. This multiplex PCR-based assay detects three ruminant species, verifies porcine origin and could be used as a screening tool or as a confirmatory assay. It is capable of providing additional assurances of crude heparin quality and will help to identify and control the species origin of the heparin supply. The control of the animal origin of crude heparin is important to ensure the safety of drugs and devices that contain heparin and to protect public health.

Introduction

FDA's Draft Guidance for Industry, titled “Heparin for Drug and Medical Device Use: Monitoring Crude Heparin for Quality,” alerts manufacturers of active pharmaceutical ingredients (APIs), pharmaceutical and medical device manufacturers of finished products, re-packers, and others, to the potential risk of crude heparin contamination.

This draft guidance provides recommendations that will help API manufacturers, pharmaceutical and medical device manufacturers of finished products, repackers, and others, to better control their use of crude heparin that might contain oversulfated chondroitin sulfate (OSCS) or non-porcine material (especially ruminant material) contaminants. Among other things, the draft guidance recommends that drug or medical device manufacturers who receive and use crude heparin to manufacture drugs and medical devices containing heparin test and confirm the species origin of crude heparin in each shipment before use in the manufacture or preparation of a drug or medical device containing heparin. The test method should be qualified for use in testing crude heparin to verify the species of origin. The method should be based on good scientific principles (e.g., sufficient accuracy and specificity) and possess a level of sensitivity commensurate with the current state of scientific knowledge and risk. The following method is an assay method for measuring ruminant contamination in crude heparin using real-time polymerase chain reaction (PCR, hMRTA). This method has been evaluated for suitability using crude heparin of porcine origin and bovine reference materials. The method can be used to identify ruminant DNA in porcine-derived crude heparin. This protocol provides details of the method, reagents and equipment required to execute the real-time PCR assay for the detection of ruminant material in porcine crude heparin. An appropriate alternative method or methods can also be qualified for use in screening crude heparin for the presence of ruminant material.

MATERIALS AND METHODS

It is recommended that a negative template control is run (extraction, clean-up, and PCR) with each set of samples analyzed. Additionally, commercially available genomic DNA (bovine, porcine, caprine, and ovine) should be run as a positive control on each lot of BioGX Ruminant and Porcine Beads upon receipt.

1. DNA Extraction

Introduction:

Instructions for isolating DNA from 0.5 ml of dry crude heparin are described below. Read this entire SOP before starting the purification procedure. This method uses the ChargeSwitch® gDNA Rendered Meat Purification Kit by Invitrogen (Carlsbad, CA). All reagents, pipette tips, and microcentrifuge tubes should be DNase free. Tips should also be aerosol resistant tips, reducing the chance for cross contamination at all steps of the method, from DNA extraction to PCR amplification. Because of the dry powder matrix it is recommended that procedure step 1.2.a is performed under a hood in a separate location from the DNA extraction, clean-up and PCR.

Note: PCR testing of crude heparin should be performed prior to any treatment (e.g., chemical oxidation) that could compromise the integrity of the DNA in the sample such that PCR species identification would be compromised.

1.1. Required Materials:

Kits:

ChargeSwitch® gDNA Rendered Meat Purification Kit (Product #CS400-100)
PowerClean® DNA Clean-Up Kit (Product# 12877-50)
BioGX Ruminant and Porcine Beads (Product # 204-0002)

Equipment Needed:

Micro centrifuge
Heat Block
Nuclease Free Water
Vortex
Invitrogen Magna Rack
2.0 mL Tubes
Smart-Cycler
Smart Cycler centrifuge
Smart Cycler cooling block
SmartCycler 25 µL reaction tubes

1.2. Procedure
  1. Add a well-mixed heparin sample up to the 0.5 mL sample line of the 2 mL microcentrifuge tube.

    *Critical Step: Aliquot only one sample at a time and DO NOT open the next microcentrifuge tube and source heparin sample until prior sample is completely measured.

  2. Add 1 mL of ChargeSwitch Lysis Buffer to sample. Tip the tube at a slight angle while filling the tube with lysis buffer.
  3. Add 100 µL of ChargeSwitch SDS to each tube. Vortex the tube for 5 seconds.
  4. Incubate at 95°C (water bath or heat block) for 5 minutes.
  5. Add 400 µL of ChargeSwitch Precipitation Buffer (N5) to the lysate. Vortex the tube for 5 seconds.
  6. Place the tubes on ice for 5 minutes to precipitate proteins.
  7. Centrifuge the tube at 16,100 x g for 5 minutes at room temperature to pellet the debris.
  8. Transfer 1200 µL of supernatant from the tube to a new 2 mL microcentrifuge tube.
  9. Vortex the tube containing the ChargeSwitch Magnetic Beads to fully re-suspend the beads in the storage buffer.
  10. Add 200 µL of ChargeSwitch Detergent to the tube of lysate.
  11. Add 40 µL of resuspended ChargeSwitch Magnetic Beads.
  12. Mix gently by pipetting up and down 5 times.
  13. Incubate the tube at room temperature for 1 minute.
  14. Place the tube on the MagnaRack until ChargeSwitch Magnetic Beads. have formed a tight pellet and the supernatant has cleared (approximately 1 minute)
  15. Without removing the tube from the magnet, carefully aspirate and discard the supernatant without disturbing the bead pellet. When aspirating, angle the pipette tip so that it is pointed away from the pellet.
  16. Remove the tube from the magnet.
  17. Add 1 mL of ChargeSwitch Wash Buffer to the tube and mix gently by pipetting up and down 5 times.
  18. Place the tube on the magnet for approximately 1 minute until the beads have formed a pellet and the supernatant is clear.
  19. Without removing the tube from the magnet, carefully aspirate and discard the supernatant without disturbing the bead pellet. When aspirating, angle the pipette tip so that it is pointed away from the pellet.
  20. Remove the tube from the magnet.
  21. Add 750 µL of ChargeSwitch Wash Buffer to the tube and mix gently by pipetting up and down 5 times.
  22. Place tube on the magnet for approximately 1 minute until the beads have formed a pellet and the supernatant is clear.
  23. Without removing the tube from the magnet, carefully aspirate and discard the supernatant without disturbing the bead pellet. When aspirating, angle the pipette tip so that it is pointed away from the pellet.
  24. Add 750 µL of ChargeSwitch Wash Buffer to the tube and mix gently by pipetting up and down 5 times.
  25. Place tube on the magnet for approximately 1 minute until the beads have formed a pellet and the supernatant is clear.
  26. Without removing the tube from the magnet, carefully aspirate and discard the supernatant without disturbing the bead pellet. Remove all the supernatant after the final wash.
  27. Remove the tube containing the pelleted magnetic beads from the magnet. There should be no supernatant in the tube.
  28. Add 75 µL of ChargeSwitch Elution Buffer (E5) to the tube.
  29. Pipet up and down gently 10 times using an adjustable pipette to resuspend the ChargeSwitch Magnetic Beads.
  30. Incubate at room temperature for 1 minute.
  31. Place the tube on the magnet for 1 minute until the beads have formed a tight pellet and the supernatant is clear.
  32. Without removing the tube from the magnet, carefully transfer the supernatant containing the DNA to a new, sterile 2 mL microcentrifuge tube without disturbing the pellet. When pipetting, angle the pipette tip so that it is pointed away from the pellet.
  33. Discard the used ChargeSwitch Magnetic Beads.

    * DNA should be stored at -20°C or continue to DNA Clean-up

2. DNA Clean-Up

Introduction:

Instructions for removing PCR inhibitors (e.g., heparin) from previously isolated DNA from crude heparin are described below. Read this entire SOP before starting the clean-up procedure. This method uses the PowerClean® DNA Clean-Up Kit by MoBio Laboratories (Carlsbad, CA). All reagents and microcentrifuge tubes are DNase free and provided by the company.

2.1. Procedure
  1. Add 75 µL of nuclease free water to the DNA sample.
  2. Add 70 µL of PowerClean DNA Solution 1 to DNA. Gently invert 3-5 times to mix.
  3. Add 20 µL of PowerClean DNA Solution 2 and invert 3-5 times to mix.

    Note: Check PowerClean DNA Solution 2. If it has precipitated, heat to 60°C and gently invert the tube periodically until it has completely dissolved. Do not vigorously shake as this may result in foaming. This solution may be used while still warm.

  4. Add 85 µL of PowerClean DNA Solution 3 and invert 3-5 times to mix. Incubate 4°C for 5 minutes.
  5. Centrifuge tubes at 10,000 x g for 1 minute at room temperature.
  6. Avoiding pellet, transfer the entire supernatant into a clean 2 mL Collection Tube (provided).
  7. Add 70 µL of PowerClean DNA Solution 4 and invert 3-5 times to mix. Incubate at 4°C for 5 minutes.
  8. Centrifuge tubes at 10,000 x g for 1 minute at room temperature.
  9. Avoiding pellet, transfer the supernatant into a clean 2 mL collection tube (provided)
  10. Shake to mix PowerClean DNA Solution 5. Add 800 µL of PowerClean DNA Solution 5 to the supernatant and vortex for 5 seconds.
  11. Load approximately 600 µL of the supernatant onto spin filter and centrifuge at 10,000 x g for 1 minute at room temperature into a clean 2 mL Collection Tube (provided)
  12. Discard flow through. Add remaining 600 µL supernatant to spin filter and centrifuge at 10,000 x g for 1 minute at room temperature.
  13. Discard flow through. Add 500 µL of PowerClean DNA Solution 6 to spin filter and centrifuge at 10,000 x g for 30 seconds at room temperature.
  14. Discard flow through.
  15. Centrifuge spin filter at 13,000 x g for 2 minutes at room temperature.
  16. Carefully place spin filter in new 2 mL collection tube (provided). Avoid splashing any PowerClean DNA Solution 6 onto spin filter.
  17. Add 75 µL of PowerClean DNA Solution 7 to center of white filter membrane.
  18. Centrifuge at 10,000 x g for 30 seconds at room temperature.
  19. Discard spin filter. Store at -20°C or continue to PCR amplification.
3. Real-Time PCR Amplification

This procedure is designed to analyze presence of ruminant DNA in porcine-derived heparin samples using the SmartCycler from Cepheid. Using other platforms will require the appropriate modification to validate this portion of the SOP.

3.1. Real-Time Assay Preparation
  1. Remove re-sealable pouch of Sample-Ready Beads from refrigerator. Open pouch by tearing off the sealed area at the top of the pouch at the notches.
  2. Remove as many tubes as needed from the pouch and gently open each tube.
  3. Add 25 µL of nuclease free water to each tube, tip mix, and close tube lids.
  4. Using a microcentrifuge, quickly spin all tubes (5 seconds).
  5. A bead blank (bead +water/no DNA) must be analyzed with each set of samples analyzed.
3.2. Procedure
  1. Aliquot 25 µL of the previously prepared supermix (bead and water) to a SmartCycler PCR reaction tube.
  2. Add 1 µL of sample to each corresponding PCR reaction tube.
  3. Cap the tubes and spin in the SmartCycler centrifuge (5 seconds).
  4. Place the PCR tube in each well of the SmartCycler block and close individual lids.
  5. Prepare Run Program:
  6. Click Create Run Icon. Select Dye Set FCTC.
  7. Select protocol (see section 3.3).
  8. Select appropriate number of sites (i.e., number of PCR tubes in run).
  9. Give each site a Sample ID according to components in each tube.
  10. Click Start Run.

    Note: A positive result requires a cycle threshold (Ct) value.

3.3. PCR Cycling Conditions

This protocol must be set prior to beginning a PCR run. Save parameters using a unique name.

PCR Program:

Stage 1: 95.0°C for 120 seconds (optics off)

Stage 2: 45 cycles

     95.0°C for 10 seconds (optics off)
     56.0°C for 60 seconds (optics on)

Note: The Ct value for analysis should be set at the default value of 30.

4. Interpretation of Results

The FCTC dye set is used for fluorescence monitoring. A positive result for ruminant DNA consists of a sample having a Ct value in the FAM channel before 45 cycles. A positive result for porcine material consists of a positive Ct value found in the TxR channel. The Internal Amplification Control (IAC) reports in the Cy5 channel and helps to mitigate false negative reporting. Many different heparin samples types contain PCR inhibitors. This test includes an IAC to ensure conditions are favorable for PCR, thus minimizing the reporting of false-negative results due to inhibition of the hot start polymerase. Specifically, the IAC is included to determine if PCR inhibition is present in samples that report a negative result. The IAC primers and probe target a synthetic sequence that is included in the reaction mix.

When no PCR inhibition is present from the sample and in the absence of amplification of targets, the IAC should produce signal with a Ct value between 32 and 37 cycles. If the sample targets are present at high concentrations, the IAC may or may not report amplification due to competition. This is normal.

If the IAC is delayed beyond a Ct value of 37 or does not report at all in the absence of target amplification, the samples may contain PCR inhibitors that are preventing target detection. If this event is observed, a re-extraction and additional clean-up using a new aliquot of the stock crude heparin sample will be required.

Ruminant positive sample results should be confirmed by testing the same DNA sample by PCR two additional times where all three test results are positive.

CONCLUSIONS

This method presents a protocol to perform a rapid multiplex real-time assay for the detection of ruminant material in porcine-derived crude heparin. The evaluation of the hMRTA included an assessment of the specificity, sensitivity, and selectivity. An additional trial of the hMRTA was successfully conducted, in a blinded manner, by an external laboratory for peer verification. The hMRTA successfully passed the in-house evaluation as well as the external peer verification. The hMRTA accurately detects 0.5% (wt. /wt. basis) ruminant (cow, sheep, and goat) material in porcine crude heparin, as well verifies porcine origin of the crude heparin.

Page Last Updated: 06/16/2014
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