Pocket tips: shaking and breaking
This is the second article in the series designed to help you improve results from your Pocket Diagnostic tests. In the first article we discussed sample selection. This time, we take a look at why sample preparation is so important.
How well you have prepared the sample is a key factor in diagnosing disease, whatever the test method. Here are some tips for making sure you carry out sample preparation to best effect when using Pocket Diagnostic tests.
There is no doubt about it, careful sample preparation helps improve the performance of on-site tests. This is not surprising when we consider what is happening during the sample preparation stage of a Pocket Diagnostic test.
Preparing a sample has two main functions.
• Firstly, separating the pathogen from the plant material so that it can be detected by the test, and
• Secondly ensuring that the sample is sufficiently fluid to flow along the membrane in the test strip carrying the target pathogen.
Keeping these two functions in mind is helpful when samples are being prepared.
Sample preparation
Before we take a closer look at preparing samples from different types of plant tissues, let us consider the way in which samples are prepared. Pocket Diagnostic tests use the unique ‘bottle and ball’ extraction method. Samples of plant to be tested are put into the small bottle containing 5ml of buffer and shaken. Five stainless steel ball bearings in the bottle break up the plant tissue during the shaking phase. Shaking thoroughly is important.
For the test to detect pathogen, it must be released from the plant cells. For example, in order to detect a virus inside plant cells, those cells must be broken open to release the virus particles. Only then can the antibody on the test strip ‘capture’ the pathogen to make the positive ‘T’ line. The same principle applies also to fungal and bacterial pathogens.
Helpful tip: vigorous shaking is best. Shaking briefly, or not shaking hard enough, can fail to release pathogen from tissues.
Now let’s consider viscosity. For the test to function correctly, the sample must flow along the membrane in the test stip. If too much material is suspended in the buffer then the pores in the membrane will block, causing the test to fail. Therefore, putting too much plant material in the extraction bottle can cause the test to fail simply through the amount of suspended solids being applied to the test strip.
Helpful hint: don’t be tempted to add too much plant material to the extraction bottle ‘just for good measure’. Aim to use 0.2g of symptomatic sample, which equals approximately 25 x 25mm of ‘typical’ leaf tissue.
Sample types
Pocket Diagnostic tests can be used on a wide variety of sample types, but to get the best out of sample preparation steps, here are a few rules of thumb:
• Soft tissues such as tomato leaves – 20 seconds of shaking should be sufficient
• Thickened, or waxy leaves such as Rhododendron - cut, tear or score the leaf samples before adding to extraction bottle. Shake for at least 30 seconds and up to one minute depending on tissue
• Stem tissue (non-woody) – cut into small pieces and shake for at least 30 seconds.
• Roots – rinse with water to remove soil or compost. Pull off small samples of thin roots and shake for 30 seconds. If roots are larger, takes slices of the infected area and shake for up to one minute
• Woody samples – take slivers of symptomatic tissue and cut into small pieces before shaking in the extraction bottle for at least one minute.
• Fruit tissues – where possible, use the skin or outer tissues as samples. Internal fleshy tissues (eg strawberry or tomato) tend to block the membrane.
If you have any questions on sample preparation for the types of tissue you are dealing with, please let us know. Our technical experts will help you get the most out of the tests by improving your sample preparation.