Test Methods Demystified

Five things to keep in mind about testing

1. The sample is not the whole

Good sampling protocols use science and statistics to provide a high probability of disease detection but there is still a chance that a test of an infected individual or population returns a negative result. This is because we seldom test an entire organism or population. We typically sample parts of plants and those only from a subset of the plant population.

Successful disease control will reduce infections, and all else being equal, increase the sample size needed to detect infection in the population.

2. No test is perfect

The performance of a test can be judged by its sensitivity and specificity. Sensitivity is the ability of the test to detect very low levels of the target pathogen. Specificity is the ability of the test to differentiate its target from similar pathogens. The perfect test would be 100% sensitive and 100% specific – but there are no perfect tests in the real world.

As a rule of thumb, the greater the sensitivity of a test, the greater the risk of a false positive, and the greater the specificity, the greater the risk of a false negative.

3. Pathogens can change

Pathogens such as viruses mutate over time and new strains replace older ones. A test that worked for an older strain will not always perform as well on a new strain. Like the antivirus software on your computer, tests for plant pathogens need regular updating.

4. You only find what you look for

Except for high-throughput sequencing, all the tests discussed below will only detect the pathogen they were designed to detect. For example, trees that tested free of the six viruses covered by the Deciduous Fruit Plant Certification Scheme were recently found to be infected with the viroid that causes plum marbling. The trees had not previously been tested for plum viroid 1 because no one knew it existed.

Plum viroid 1 is now included in the Scheme, but it is unlikely that this will be the last time that a new pathogen emerges in fruit trees.

5. Things can go wrong

Like any undertaking, testing is subject to human error. The best way to mitigate this risk is to use reputable laboratories that have preferably been accredited by SANAS – the South African National Accreditation System.

What do tests detect?

The tests discussed below are used in SA to detect viruses and viroids. There are six viruses and one viroid covered by the Deciduous Fruit Plant Certification Scheme. They are included in the Scheme because they occur in SA and are economically important.

Many other significant viruses and viroids do not occur in SA – testing imported plant material helps to keep them out.

Viruses all consist of genetic material enclosed in a protein coat. Tests for these viruses are designed to detect either their genetic material or their proteins. Viroids do not have a protein coat, so tests for viroids only target genetic material.

Why does this matter? Because it explains a key difference between ELISA and PCR. ELISA tests are based on viral proteins while PCR tests sniff out viral or viroid genetic material. LAMP assays and high-throughput sequencing likewise target genetic material.

None of these technologies is used exclusively for finding viroids and viruses in fruit trees. They are applied daily throughout the world to test a wide range of biological samples for both diagnostic and research purposes.

ELISA

ELISA is short for enzyme-linked immuno-sorbent assay, a laboratory workhorse that has been around for more than 50 years. ELISA testing is available for apple chlorotic leaf spot virus, apple mosaic virus, apple stem grooving virus, prune dwarf virus, and Prunus necrotic ringspot virus.

ELISA relies on the ability of antibodies to bind to antigens. Antibodies are proteins produced by the immune system. These proteins recognise and help inactivate foreign material such as viruses or bacteria in the body.
Antigens are defined in relation to antibodies – an antigen is something to which an antibody binds. Certain immune cells also bind antigens.

The key to understanding the ELISA is knowing that antibodies are specific. An antibody will only bind to a specific antigen in the same way as a key will only open a specific lock. Scientists can manufacture antibodies to specific viral proteins and then incorporate those antibodies in an ELISA to detect the virus in a sample.

There are umpteen variations of the basic ELISA, but the fundamentals are the same for all of them. Figure 2 shows how a double antibody sandwich ELISA works. This is a method commonly used in SA to test for plant viruses.

PCR

The invention of the polymerase chain reaction – PCR – nearly 40 years ago was a seismic event in biology. PCR is behind everything from forensics in television shows like CSI to schemes to revive the extinct woolly mammoth.

Closer to home, PCR testing is used for apple chlorotic leaf spot virus, apple mosaic virus, apple stem grooving virus, apple stem pitting virus, prune dwarf virus, Prunus necrotic ringspot virus, and plum viroid 1.
PCR works because DNA usually consists of two complementary strands.

Either strand can serve as a template to recreate the other strand. The PCR process splits double-stranded DNA and then recreates two copies of the original DNA from the two strands. Repeat the cycle once and two copies become four copies. Repeat the cycle 30 times and you end up with 1 billion copies – this is called amplification.

The reason PCR is so useful is that one can control which DNA is amplified. For example, if you want to test a plant sample for apple mosaic virus, you can design a PCR to only target that virus and not any other viruses or DNA from the plant itself.

Dealing with targets that have RNA instead of DNA – such as many viruses – requires first converting RNA to DNA. Reverse-transcription PCR – RT-PCR for short – refers to PCR that includes this step. See figure 3 for more details.

LAMP

The principles of DNA amplification are the same for LAMP – loop-mediated isothermal amplification – as for PCR, except that a different strategy for amplification is employed. At present, LAMP testing is only available for plum viroid 1 in SA.

Coloured pH indicators or the incorporation of fluorescent dyes can be used to detect LAMP-amplified DNA. The pH-dependent colour change can be seen by the eye, while the dye-based tests generally require instruments, some of which are basic and others that are sophisticated – and pricey.

One advantage of LAMP over PCR is that sample preparation for LAMP is simpler, which reduces cost.

High-throughput sequencing

Also known as next-generation sequencing, high-throughput sequencing – HTS – is a method for determining the sequence of the bases in a DNA molecule. DNA has four bases – think of it as an alphabet with only four letters – that spell out the instructions for making an organism. The instructions for building a human are obviously different from those for building a pear tree and this is reflected in their genome sequences.

Genome sequencing has many practical uses. For example, potential pathogens can be smoked out by sequencing samples from diseased and healthy individuals and seeing what genetic material is present in the former but not in the latter. This is how plum viroid 1 was identified.

High-throughput sequencing differs from most other tests in being non-specific. Whereas PCR will only detect what it was designed to detect, HTS can potentially detect previously unknown things – like plum viroid 1.

The drawback of HTS is that interpreting the results can be challenging and requires expert analyses of reference sequence information that may not be available. High-throughput sequencing is also an expensive and time-consuming process. This makes it unsuitable for testing large numbers of samples.