The practice of using botanicals to improve health dates back thousands of years. Recently, increased demand, coupled with unsustainable harvesting practices and climate change, have placed a stress on the supply chain. Because of this, botanical adulteration is becoming a growing concern, making botanical identification an increasingly essential part of quality control.
Ideally, identification is performed as close to the source as possible, so resources can be efficiently allocated to the material of correct identity. There are a number of approaches for botanical identification. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods.
Morphological identification is based on differences in macroscopic and microscopic features of plant materials. However, it requires a well-trained botanist and its application to powdered botanical materials is limited. Chemical analytical methods are widely used in pharmacopoeias and laboratories, but are not ideal for field testing due to the size of instruments and environment requirements.
Recently, genomic methods have emerged as an alternative technique for botanical species identification due to the high fidelity and specificity of genomic information in plant materials. With molecular diagnostic tools now available in a form of portable devices, this approach becomes possible for field application. The goal of this protocol is to introduce a method for botanical identification in situations where access to the laboratory equipment and expertise is limited, such as the farm supplying the botanical material, using a portable qPCR system.
Matricaria chamomilla, commonly known as German chamomile, has been used as an herbal medication to promote health for centuries. To demonstrate the specificity of the current method, the differentiation of German chamomile from another commonly used chamomile flower, known as Roman chamomile, is included for comparison. The field identification procedure is demonstrated in the middle of a German chamomile farm.
Set up a testing area in the field with a flat and horizontal surface. Identify a representative plant that reflects the characteristics of the majority of plants in the chamomile flower field. Pick a flower head from the representative plant using sterile gloves.
Place the sample into a 2.0 milliliter collection tube. Repeat and collect a leaflet approximately 0.5 to 0.7 centimeters long from the same plant. Preheat the dry bath incubator to 95 degrees Celsius.
To each collection tube, add 100 microliters of the extraction solution from the plant DNA extraction kit. For better DNA extraction efficiency, grind the sample using a tissue pestle. Close the tube.
Ensure that the botanical tissue is covered by the extraction solution throughout the extraction process. Place the collection tubes in a preheated dry bath incubator, and incubate the collection tubes at 95 degrees Celsius for 10 minutes After 10 minutes, take the tubes out of the dry bath incubator. Add 100 microliters of the dilution solution from the DNA extraction kit and mix the solution by pipetting up and down several times.
Repeat the same step for leaflet extraction. Shake to mix the solution further. The plant issue usually does not appear to be degraded after this treatment.
The liquid color may change and become cloudy. Configure the qPCR instrument thermocycling conditions according to table one, which starts with a constant temperature step for initial denaturation, followed by 25 cycles of amplification and ends with temperature ramping to obtain a high-resolution melting curve. Thaw the qPCR master mix and primers listed in table two at room temperature prior to use.
Plan the reaction that will be loaded in each well. Wells containing positive controls with the target species, positive controls with non-target species, samples and negative controls. In this example, 10 wells are used, five for the German chamomile identification test and another five for the Roman chamomile identification test.
For each type of species identification test, one well contains a positive control with DNA extracted from the targeted species reference material, one well contains a positive control with DNA extracted from non-targeted species reference material, two wells are filled with flower and leaf DNA samples extracted from the field, and one well is allocated for a negative control. Table three describes each well type. Prepare a reaction master mix according to table four for each botanical species identification test.
A typical reaction master mix contains universal qPCR master mix, two times, forward and reverse species specific primers, and nuclease-free water. Thoroughly mixed the reaction master mix by pipetting before use. Place the PCR reaction cartridge face up on a flat and stable surface.
Load 18 microliters of the reaction master mix configured in the previous step into the cartridge wells according to the wells defined here. For this demonstration, add the German chamomile identification test reaction master mix into wells labeled for GC test, GCT in wells one, three, five, seven, nine, and the Roman chamomile identification test reaction master mix into wells labeled for RC test, RCT in wells two, four, six, eight, 10. Transfer two microliters of sample DNA from the supernatant of the DNA extraction tubes and pre-extracted DNA positive controls into cartridge wells preloaded with qPCR master mix.
After adding each DNA template to the qPCR master mix, gently mix the solution by pipetting. Carefully seal the cartridge with adhesive film. Load the cartridge onto the thermocycling chamber and close it.
Set the qPCR instrument to run. In the current protocol, intercalating dye is used to measure the amplification of target fragments in real-time. The Ct value is defined as the number of cycles required for the fluorescent signal to cross the predefined threshold.
A Ct value less than 25 cycles was considered as positive amplification. In this figure, the Roman positive control was only amplified in the Roman chamomile identification test, but not in the German chamomile identification test. The German chamomile positive control and field samples were only amplified in the German chamomile identification test, but not in the Roman chamomile identification test.
The negative control was not amplified in either test. To further confirm specific amplification in positive controls and samples, fractions of PCR and products from each well were run on 2%agarose gel in the laboratory. All wells with a Ct value less than 25 yielded amplicons at their expected sizes, which differ between German chamomile and Roman chamomile.
The rest of the lanes showed no specific amplification product, in agreement with the absence of fluorescent signal for these wells as observed in field testing. Following PCR amplification, a melting curve analysis was performed using the same software that was used to monitor the fluorescent signal. With the increase of temperature, the double-strand amplicons begin to dissociate, resulting in the decrease of fluorescence intensity.
The fluorescence intensity changes were further converted into melting peak curves for further differentiation of German chamomile from Roman chamomile by their characteristic melting peaks. In this figure, the melting peak of the German chamomile positive control is 85.6 degrees Celsius and it is distinct from the melting peak of the Roman chamomile positive control. The PCR amplicons from field samples produce melting peaks close to the German chamomile positive control.
The identity of the field sample is determined based on specific amplification and characteristic melting temperature that matches controls. In this example, both flower and leaf collected in the field are identified as Matricaria chamomilla, German chamomile. Roman chamomile test results for these two samples are negative.
The protocol demonstrates the identification of German chamomile in the field using a portable qPCR system. Similar methods can we develop to expand the portfolio of botanicals that can be tested. We hope this video will provide valuable training materials for scientists, even non-experts to perform botanical identification in the field or in an environment with limited laboratory equipment.
DNA-based field identification testing not only produces highly accurate results that match laboratory analysis, but also reduce the time and costs associated with traditional methods performed in a laboratory.
