PCR test for detection of M. bovis in post-mortem tissue samples

The Animal & Plant Health Agency (APHA) plays a central role in delivering the bovine tuberculosis (bTB) eradication programmes in England and Wales and the bTB surveillance programme in Scotland. Diagnostic and confirmatory testing is the cornerstone of any disease eradication programme and APHA is responsible for all the laboratory based bTB testing to support this effort. 

Until recently the “gold standard” for bTB diagnostic testing from tissue samples (both bovine and non-bovine farmed animals) was traditional microbiological culture. Although well established, the major disadvantage of this approach is the length of time (6-22 weeks) it typically takes to obtain a result.

APHA has validated the polymerase chain reaction (PCR) test which can detect the bacterium responsible for bovine TB, Mycobacterium bovis (M. bovis) directly from tissue samples collected at post-mortem inspection. The major advantage of this method is that it typically takes only three weeks to report a result to the keeper.

The PCR test became operational on 30 March 2022 and was initially only being used in Great Britain (GB) for tissue samples from carcases of:

  • TB slaughterhouse cases in cattle and non-bovines i.e. animals routinely sent for private commercial slaughter that have suspicious lesions of TB at routine meat inspection;
  • Non-bovine animals such as camelids, goats, pigs, sheep and farmed deer that are removed as TB test reactors, direct contacts or clinical TB suspects, and cases where TB lesions are identified on diagnostic post-mortem examination in a veterinary laboratory; and
  • Domestic pets (cats and dogs) and exotic species of animals (e.g. in zoological collections) submitted to APHA for laboratory investigation.

After a period of operation and review of the test’s performance, on 14 February 2024 APHA extended the use of the PCR test to tissue samples from post-mortem samples taken from TB test positive cattle (skin, gamma, etc), direct contacts (DC) and compulsory or privately slaughtered/dead Inconclusive Reactors (IR).

The PCR test reduces the time taken by the APHA laboratory to confirm whether M. bovis is present in tissue samples taken from carcases at post-mortem inspection. This means that in certain situations (i.e. slaughterhouse cases), if the PCR test results are negative, and check testing has been completed where required, APHA can lift herd movement restrictions sooner than the previous protocols using microbiological culture, which typically take 6-22 weeks. If the PCR test results are positive, APHA can inform the keeper and their private vet quickly, so that further action can be taken as soon as possible to control TB in the affected herd.


Polymerase chain reaction (PCR) is a molecular-based laboratory technique that involves detecting small amounts of the genetic material (DNA) that is contained within all living organisms. This is done in the laboratory by making millions of copies of a specific sequence of the target DNA, a process called ‘amplification’.

Each M. bovis bacterium contains unique DNA which carries the genetic instructions for its development, function, growth and reproduction. PCR can detect tiny amounts of the DNA present within these bacteria and amplify it to produce a quantity which is then detectable. By looking for targeted bacterial DNA, the APHA laboratory is able to rapidly detect and differentiate the presence of M. bovis bacteria in tissue samples taken from carcases at post-mortem inspection. Critically, this detection is significantly quicker than the microbiological culture method.  

PCR test results are typically available within three weeks. Depending on the sample type submitted, traditional culture can take 6-22 weeks for confirmation.

No. The PCR test is designed to be used on tissue samples taken from the animal’s carcase at post-mortem examination or meat inspection. Exception from this are fresh biopsy samples collected from live domestic pet mammals (e.g. cats/dogs) suspected of being infected with M. bovis. The PCR is not tissue- or organ-specific and is applicable irrespective of the anatomical location of the TB lesions. PCR is not currently validated for use on badger carcases, fixed biopsy tissue or bodily fluids.

Yes. APHA requires all new diagnostic tests to be fully validated in accordance with World Organisation for Animal Health (WOAH) guidelines to provide assurance in the results generated. Validation involves multiple steps to assess the diagnostic performance characteristics of the test and show that it is specific (it only detects the organism of choice) and it is sensitive (it will have a high probability of detecting the organism if present). 

In a validation study carried out by APHA, the M. bovis PCR test has been shown to produce equivalent results to the traditional microbiological culture method, for both bovine and non-bovine tissue samples.

In a very small proportion of cases it may not be possible to obtain a valid PCR test result. In those instances, APHA will use bacteriological culture to attempt to grow the TB bacterium. APHA will inform keepers if this applies to samples from their herd/flock.

The PCR test is targeting two different DNA sequences. One of them is found in all mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC) (which includes M. microti, M caprae and M. tuberculosis in addition to M. bovis) and the other one is specific to M. bovis. To be considered positive, a sample must be positive for both targets. Occasionally, a sample may be found to be positive for MTBC but negative for M. bovis. This may happen because other mycobacteria from the MTBC is involved, or because the amount of M. bovis DNA in the sample is very low. Such samples, similarly to those where the PCR test fails to produce a valid result, are subjected to the traditional culture method to obtain a final result. Once the culture process is completed there are a few possible outcomes. If no growth is achieved the sample is reported as negative for M. bovis. If growth is achieved the sample goes through WGS analysis. Occasionally M. bovis may still be identified at this stage and ultimately the sample will be reported as positive. The detection of any other mycobacteria will be reported as M. bovis negative.

The main benefit is rapid detection of M. bovis directly from post-mortem tissue samples. PCR test results are reported to the keeper typically within three weeks. This is quicker than the gold standard of microbiological culture to grow the M. bovis bacterium in the laboratory, which typically takes 6-22 weeks for a result. Rapid test results have significant advantages where a quicker diagnosis allows more effective case management, for example slaughterhouse cases or TB breakdowns where only reactors with no visible lesions were detected but subsequently some of them are found to be PCR positive.

Another benefit is that the cost of a PCR test per tissue sample is lower than that of microbiological culture. The process is semi-automated and exploits laboratory robotics to facilitate high throughput, whilst also minimising contamination and maximising traceability of samples.

The one important limitation of this test is that it is not possible to apply whole genome sequencing (WGS) directly on the M. bovis target DNA sequence that is amplified through PCR testing (i.e. the PCR product). WGS is only possible with DNA obtained from a pure microbial culture. WGS is a molecular technique that characterises the entire DNA content of an M. bovis isolate. This technique has replaced genotyping, which only looked at certain sections of the bacterium’s DNA. WGS allows differentiation of the ‘strains’ of the M. bovis bacterium at greater resolution than genotyping. WGS is now routinely carried out by APHA on M. bovis isolates from TB breakdown herds to help identify the probable source of infection. APHA cannot rely exclusively on the PCR test for case management and epidemiological analyses of TB breakdowns as culture still has to be carried out for WGS to be performed. Culture is only carried out where WGS is required to allow full analyses of the breakdown and identify the probable source of infection. APHA will not report culture results unless they are used for the confirmation or negation of M. bovis infection i.e. in the rare instances where the PCR test has not provided a valid result. The additional WGS analysis of positive PCR samples will not interfere with or delay APHA’s decisions to manage the TB breakdown.

A slaughterhouse case means that typical lesions of TB have been unexpectedly found at routine meat inspection of the carcase of a bovine animal sent for private commercial slaughter. When a slaughterhouse case is identified, the herd of origin’s officially TB free status is suspended (OTFS), which means that bovines can’t move on or off the holding except under a licence issued by APHA. Previously, tissue samples were taken from the carcase and submitted for microbiological culture which can take up to 22 weeks for a result. In the meantime, the herd remained under movement restrictions and a skin test (check test) of the herd may have been carried out pending the final culture result.

Now that PCR testing is operational, instead of relying on culture results for confirmation or negation of M. bovis infection, PCR testing is carried out on the lesions sampled from the slaughterhouse case carcases. PCR test results are typically reported to the keeper within three weeks and, if negative, herd movement restrictions could be lifted sooner instead of waiting for culture results. This limits the negative impact of movement restrictions on cattle herds and (in England and Scotland only) in most cases, eliminates the need for a check test. PCR test positive results allow for more timely deployment of additional disease control measures, such as source or spread tracing and application of interferon-gamma testing where applicable.

In England and Scotland, the shorter turnaround time of the PCR test results will allow for breakdowns triggered by non-visibly lesioned (NVL) Reactors (Rs)/Direct Contacts (DCs)/Inconclusive Reactors (IRs) which are then PCR test-positive to be re-classified from Officially Tuberculosis Free Suspended to  (OTFS) to Officially Tuberculosis Free Withdrawn  (OTFW) sooner. This will subsequently allow for timelier deployment of the enhanced surveillance (i.e. tracing, contiguous or radial testing) and case management (i.e. the use of IFN-γ test) measures. TB breakdown herds in which at least one R/DC/IR is found to have visible lesions (VLs) at post-mortem examination have their OTF status withdrawn (OTFW) on this basis alone. Therefore, the PCR test result will have no impact on the management of such cases and the established policy procedures will continue to be followed.

In Wales all breakdowns triggered by test positive cattle are considered OTFW by default so the PCR test result will have no impact on the case management.

The PCR test allows rapid detection of M. bovis in tissue samples from carcases of non-bovine animals to confirm or negate TB infection in suspected cases of TB identified at post-mortem examination in the laboratory, or during routine post-mortem meat inspection in the slaughterhouse.

When APHA receives notification from the Food Standards Agency or Food Standards Scotland of a slaughterhouse case of TB in a non-bovine animal, or a suspect case following post-mortem examination of an animal with clinical signs consistent with TB, movement restrictions are automatically applied to the herd/flock of origin in most cases.

Previously, samples were sent for culture and WGS if M. bovis was suspected. If M. bovis was not isolated, in the majority of cases movement restrictions were lifted without further testing. The PCR test is now used for this purpose, with positive samples that meet certain criteria being sent for culture and WGS.

PCR test results are typically reported to keepers within three weeks, which means that if the result is negative APHA may be able to lift herd movement restrictions sooner. This has particular advantages for farms on which movement restrictions have the most impact, e.g. commercial pig farms and herds that rely on regular sales of breeding and fattening stock to other farms.

In the rare cases where a valid PCR test result cannot be obtained (see above), movement restrictions will be maintained until negative culture results are received.

The PCR test is also used by APHA to detect M. bovis in non-bovine animals that have been removed as TB test reactors or direct contacts, whenever APHA considers that the procedure is necessary in addition to a post-mortem examination. In these cases, even if no typical TB lesions are identified at post-mortem examination and the PCR test result is negative, additional testing will still be required before movement restrictions can be lifted.