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NEW ZEALAND BEEKEEPER, JUNE 2017
MPI MANUKA POLLEN DNA TEST What is the background to the test? MPI’s Mānuka Pollen DNA test works on the principle that bees collecting nectar from mānuka plants will also collect some mānuka pollen at the same time, which will end up in the honey. As pollen grains contain DNA, we can use DNA testing techniques to test honey for mānuka pollen. Real Time PCR – the purified DNA has special reagents added to it, including small sequences of DNA (primers) that have been specifically designed to target DNA sequences that are unique to mānuka, and some DNA probes that have chemicals bound on to them that fluoresce. The sample is loaded onto a PCR instrument, which is pre- programmed to correctly run the test.
What results come from it, and how do I understand them? The results of an MPI mānuka pollen DNA test are reported as a ‘Cq’ value, and for a sample to meet MPI’s standard for mānuka honey, the Cq value needs to be less than 36. To understand this, a brief explanation is needed. Real Time PCR relies on things called ‘primers’ and ‘probes’ that are specific to mānuka DNA, and are added to the sample as part of the PCR reagents. When testing a sample, the PCR instrument goes through a series of cycles, and in each cycle: • The primers and probes attach to any mānuka DNA which is there; and • A copy of the targeted areas of DNA is made; so that • At the end of the cycle there are two times as many copies of the mānuka DNA than there were at the start of the cycle. For example, if there were 10 pieces of mānuka DNA in a sample at the start, there will be 20 at the end of the first cycle; 40 at the end of the second cycle, 80 at the end of the third cycle; and so on. MPI has set a threshold level of mānuka DNA that must be in a sample being tested by PCR, and the Cq value is the number of PCR cycles needed to reach that threshold. The more mānuka DNA in the sample at the start, the fewer cycles will be needed to reach the threshold. If a sample takes 36 or more cycles to reach the threshold (a Cq value of 36), then the original honey sample does not have enough DNA in it to meet MPI’s proposed mānuka definition. A Cq result of 36 is very close to the method’s reporting limit. Because of Uncertainty of Measurement at this level, we suggest that a Cq result of 34.5 or higher is treated as being at risk of failing if re-tested in future.
DNA stands for ‘deoxyribonucleic acid’, and is found in cells of every living organism (including plants like mānuka). The DNA of all plants is actually quite similar; however, different species or varieties of plants have their own unique sequences of DNA, and DNA tests are designed to look for these differences. How is it done? The pollen DNA test uses a technique called Real Time PCR, which is one of the common ways of doing DNA testing. Generally, the test requires about three days to complete from start to finish. The equipment needed is not too expensive (perhaps $100,000 in total). However, the testing process is quite expensive, with a number of manual steps and expensive consumables. Overall, labs are tending to charge $90–$100 + GST per sample. There are five main steps in the testing process: Isolate the pollen from the honey sample – some honey is weighed into a testing tube, and after being dissolved with water the sample is spun in a centrifuge. The pollen forms a pellet in the bottom of the tube as it spins, and the rest of the dissolved honey can then be poured away. Break up the pollen and release the DNA from inside it – after washing the pollen to remove any honey residues, an extraction solution is added. Among other things, it contains very small and hard micro-beads. The sample is put onto a very aggressive shaker (called a bead beater) for a few minutes, and the micro-beads bash the pollen grains and break them open. This releases their DNA into the liquid in the tube. Isolate the DNA – the liquid from the tube is poured through a special ‘affinity’ column which binds any DNA in the sample onto it, while letting the liquid and other things in the sample pass through. After a couple of washing steps, the purified DNA is washed off the column and is available for analysis.
Results are reviewed and approved for reporting – the PCR instrument will produce a result for each of the samples that have been tested. A suitably qualified member of the lab team will review these results, including quality control samples, before approving results for reporting. Quality control Laboratories carry out a number of tests and checks each time that they analyse samples for quality control purposes. In the case of DNA pollen tests, these will include: • Blank QC samples, which should have nothing in them, and therefore check for contamination in the lab. • Control samples, which are honey samples the lab uses repeatedly to check that the method is working correctly. The lab will know what results to expect for these honeys, which are usually chosen to be at the bottom and top of the normal range seen in commercial samples. • Duplicate samples, which may be the same sample tested twice in the same batch, or may be a sample from a previous batch that is re-tested. The purpose is again to confirm that the testing is producing consistent results using a ‘live’ sample submitted by a customer. • An ‘internal control’. Each sample is not only tested for mānuka DNA, but also for DNA found in all plants, to confirm that the testing process has worked for that sample. These general plant DNA tests should work well in all samples—if there is a poor result for the plant DNA markers, it will cause the lab to consider repeating the sample.
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