PCR Techniques

19.2.1 Conventional PCR

Early methods for MAP detection by PCR utilized conventional PCR (Stevenson and Sharp, 1997). In conventional PCR methods, amplification of the final DNA product only is assessed follow­ing the completion of the cycles of DNA am­plification (Lorenz, 2012).

The challenge with conventional PCR assays is the reduced analytical sensitivity compared with qPCR (Zemtsova et al., 2015; Xia et al., 2018). Nested PCR, though more sensitive, carries with it a high risk of cross­contamination due to the additional manipu­lation post-primary amplification. However, these methods are still applied in some settings, such as MAP detection in tissues from patients with Crohn's disease (Sharp et al., 2018; Zarei- Kordshouli et al., 2019). Strain typing assays still commonly use conventional PCR for IS 1311 and restriction enzyme digest to identify single nucleotide polymorphisms common to the dif­fering strains (Marsh et al., 1999; Park et al., 2018; de Albuquerque et al., 2018).

19.2.2 Real-time/Quantitative PCR

In qPCR, DNA amplification is monitored in ‘real time' during the PCR cycling by the collection of fluorescent signal data associated with the ampli­fied product. qPCR instruments measure flores­cence after each cycle, with multiple fluorescent molecules and detection channels available al­lowing for evaluation of multiple concurrent targets (multiplex). The point at which the am­plification curve crosses a definable threshold (usually set in the lower section just after the amplification curve exceeds background fluo­rescence levels) is called the quantification cycle value (Cq, also referred to as Ct) and is related to the initial copy number of the template DNA target.

Application of qPCR for the detection of pathogens is now a routine diagnostic approach used in clinical and veterinary testing and is com­monly used in paratuberculosis diagnostic testing (Alinovi et al., 2009; Park etal., 2014; Plain et al., 2014; Heuvelink et al., 2017; Arsenault et al., 2019). A broad range of assays have been devel­oped for varying sample types (see Section 19.5) with many commercial kits now also available (see Commercial Diagnostic Kits).

One of the main differences between the dif­ferent qPCR assays is the gene target and hence the primers used in the assay. Multiplex qPCR assays amplify multiple gene target-specific se­quences through the inclusion and optimization of multiple primer pairs in the same reaction mix. There are a number of multiplex assays that have been developed for the detection of MAP sequenc­es, often combined with an internal amplification control (Irenge et al., 2009; Schonenbrucher et al., 2008; Sevilla et al., 2014). These offer the poten­tial for increased specificity by targeting more than one gene from MAP.

19.2.3 Droplet digital PCR

While real-time PCR has improved timeliness and sensitivity of molecular diagnostic ap­proaches to detect MAP, the increased sensitiv­ity has come with its own problems. In closing the gap on detection of diseases with very low infectious doses or where subclinical forms of the disease exist, such as paucibacillary forms of mycobacterial infections, eliminating sample inhibition and ensuring sufficient target DNA makes it into the PCR reaction has become the limiting factor.

Review papers are now appearing in the sci­entific literature discussing the merits of ddPCR for the detection of infectious diseases (Kuypers and Jerome, 2017; Li etal., 2018) in comparison with qPCR; the advantages and disadvantages are typically described as shown in Table 19.1.

The major limitation to the introduction of ddPCR at present is the number of instrument models currently available and the cost of these instruments. This most likely explains the limited use of ddPCR in assays to diagnose paratubercu­losis; however, Ricchi et al. (2017) have success­fully demonstrated the use of ddPCR for the detection of MAP DNA. To date, ddPCR has most frequently being reported in the mycobacterial field for the diagnosis of tuberculosis (Pholwat et al., 2013; Devonshire et al., 2016; Ushio et al., 2016; Yang et al., 2017; Song et al., 2018; Yamamoto et al., 2018a; Nyaruaba et al., 2019) but more recently this has extended to leprosy. Cheng et al. (2019) reported superior detection of paucibacillary cases of leprosy in skin biop­sies using ddPCR over qPCR with sensitivities of 79.5% compared with 36.4%, respectively.

The approval by the US Food and Drug Administration of the first ddPCR-based test in 2019, monitoring the treatment of chronic my­eloid in leukaemia patients (https://www.fda.gov/ medical-devices/vitro-diagnostics/nucleic-acid- based-tests), suggests we can expect to see ongo­ing acceptance and expansion of this technology for other applications already utilizing qPCR, in­cluding those for pathogens of animals and hu­mans.

Table 19.1. Advantages and disadvantages of Droplet Digital PCR (ddPCR) vs quantitative PCR (qPCR) (adapted from Kuypers and Jerome, 2017).

availability of more ddPCR platforms will increase and the cost will come down; as this happens, the use of ddPCR in the diagnosis of paratuberculosis will undoubtedly escalate.

19.2.4 Viable MAP detection

A growing concern with molecular diagnosis of bacterial pathogens is the question, Are they alive?'. PCR-based diagnostics that produce late or high cycling threshold values are often considered to be associated with low numbers of bacteria, more commonly characteristic of subclinical or paucibacillary forms of disease. However, with diseases caused by mycobacteria, this is in itself to be expected. However, if the bacteria are not alive, how can they cause dis­ease? Traditional culture has been undertaken to confirm suspect or index cases, but this is expensive and protracted. A number of alter­native approaches have been developed to ad­dress this (Emerson et al., 2017).

Another alternative is intercalating dye­based PCR assays, where DNA from dead cells is rendered ineffective for amplification. Two dyes are commonly used: propidium monoazide (PMA) and ethidium monoazide (EMA) (Emerson et al., 2017). In both cases, compromised (dead) cells allow the dye to enter the cell and inter­calate into the DNA, thus blocking subsequent amplification, whereas intact cells do not and therefore the DNA is able to be amplified in PCR. As a result, positive PCR results are considered to be only from viable bacteria. PMA-based as­says have been described for paratuberculosis, primarily as a research tool for MAP detection in various sample types (Pribylova et al., 2012; Dalton and Hill, 2013; Ricchi etal., 2014; Kralik et al., 2010, 2014a, 2018), but have yet to be adopted more broadly. Unfortunately, these as­says contribute complexity to an already com­plex situation. Removing non-viable bacteria from a diagnostic PCR assay, where low target numbers are anticipated, will no doubt limit de­tection. This might explain the paucity of these types of assays, but this issue may be overcome with the advent of ddPCR, as previously dis­cussed. This appears to be consistent with the use of PMA-based PCR for the detection of M. tuberculosis (Mbelele et al., 2018).

19.3