Comparative evaluation of various in-house protocols on diagnostic performance for paratuberculosis IS900 PCR

Paratuberculosis is a worldwide endemic infectious disease of ruminants that results in high economic losses. Public health concerns are also being raised with human Crohn’s disease. Therefore, control is becoming priority for governments. Control is largely dependent on “Test and Cull” or “Test and Segregate” policy. Hence, it is critical to assure the infection before making the decision. Commercial kits are costly especially in view of resource limited areas. Present study analyzed the performance various in house DNA isolation methods and PCR master mix combinations to optimize a protocol for confirmation of paratuberculosis bacilli shedding in feces. Present study included five protocols of fecal DNA isolation (chemical, bio-chemical, physio-chemical and physical) and three reaction mixes (based on Qiagen, Genei and Thermo 2X master mixes) in nine different combinations using additives and tested their performance for IS900 PCR. Spiked fecal samples were used to select the best combination of DNA isolation method and PCR master mix (PRM). Selected combination was used to test reference (positive and negative) fecal samples and field samples. Findings revealed that combination physical method of DNA isolation and Genei based PRM (with additives; betaine DMSO and BSA) had lowest limit of detection. Sensitivity was 83% and specificity was 100% in comparison to fecal culture. High prevalence (23%) was reported for paratuberculosis on field samples. Optimized protocol has acceptable sensitivity and can easily be adopted in resource-limited laboratories. High prevalence of paratuberculosis needs immediate implementation of the control strategies.

Introduction effective vaccination, specific and sensitive diagnostics are required. However, lack of test for differentiating infected from vaccinated animals (DIVA) makes it difficult for preventive vaccination programs. That's why worldwide "Test and Cull" or "Test and Segregate' policy is adopted. Further, concerns also rise on the interference with bovine tuberculosis diagnosis after paratuberculosis vaccination [4]. Accurate diagnosis and counteractive action on control measures are the backbone of this strategy [5].
A wide range of diagnostic procedures are available for the diagnosis of MAP. Of these, demonstration of MAP in fecal shedding is the 'Gold Standard' [6]. Culture of feces is time consuming (8-12 weeks of incubation), costly and less sensitive [5]. PCR is a better alternative to culture and targets subspecies specific IS900 multi-copy element. However, the performance of PCR varies and depends on various factors including target, primers, DNA isolation method, PCR master mix, PCR inhibitors etc. [7]. Further, commercial DNA isolation and PCR kits are costly and are far from the affordability of resource-limited areas. Therefore, the present study aimed at investigating cost-effective, time-saving and quality DNA isolation procedure with the selection of a suitable PCR master mix (PRM) to reduce the cost and improve sensitivity. In the present study, we evaluated five DNA isolation methods and nine PCR master mixes combinations (PRMs) for their performance in IS900 fecal PCR.

Spiking of fecal samples
Fecal samples were collected from goat farm free from paratuberculosis. To ensure paratuberculosis free status; DTH skin assay, ELISA, ZN staining and fecal culture were performed. Spiking of fecal samples was done with laboratory strain of MAP. Spiking was done at 10 6, 10 4 , 10 2 , 10 CFU per gram of feces. Clinical sample collection and diagnostic testing was performed with the informed consent of farm owner.

Sample collection from paratuberculosis endemic herds
A total of n = 521 fecal samples (cattle-195, goat-110, sheep-97 and buffalo-119) were collected from field herds with history of paratuberculosis. Fecal samples were collected after the informed consent of the farm/ herd owners.
Sample collections were done during the year 2019 in the summer period (April to June). Sampling was done during the morning times (5:00-6:00 AM). Morning time was chosen because it is considered that maximum load of MAP bacilli is shed in early morning. Samples were directly collected from the rectum of animals by a trained Veterinary practitioner. These farms herds/ farms were located in District Jaipur of Rajasthan State (North-West India). All samples were transported in cold chain and were stored at -20 0 C until processing. All samples were processed for DNA isolation within one month of collection.

Methods for DNA extraction
Spiked fecal samples were subjected to DNA isolation using five methods (I-V). These methods were based on chemical, physical, biochemical and physiochemical procedures. Isolated DNA was stored at -20°C till use in PCR. Method I (magnetic nanoparticles-based DNA recovery) This method is based on the bacteria capturing magnetic nano-particles vials obtained from Genomix Molecular Diagnostics Pvt. Ltd., Hyderabad (India). One gram of fecal sample was homogenized in 10 mL of 1X PBS using pestle and mortar, homogenate was transferred to 15 mL of centrifuge tube. Tube was allowed to stand for 30 min. Supernatant was poured into a vial containing functional magnetic nano-particles and incubated for 30 min at 37 0 C. Then the vial was placed in the magnetic chamber to allow magnetic separation. Clear solution was discarded and 500 µl of PCR grade water was added and the vial was put in boiling water for 10 min and then cooled to RT. The vial was again placed in the magnetic chamber to allow magnetic separation and supernatant was collected and use as a template for PCR. Method II (biochemical) This method is based on the previous work of Singh et al. [8]. Fecal sample 1.0 g was suspended in 1.0 mL of 1X TE. To this 40 µl of lysozyme (50 mg/mL) was added and the tube was incubated for 3 h at 37 °C. Then 160 µl of 10% SDS and 40 µl of Proteinase K (10 mg/mL) were added and the mixture was incubated for 2 h at 65 °C. To this 160 µl of CTAB/NaCl was added. Contents were vortexed and incubated for 30 min at 65 °C. An equal volume of chloroform-isoamyl alcohol (24:1) was added, and the mixture was vortexed. Mixture was centrifuged at 10,000 rpm for 5 min at 4 °C and this step was repeated using the upper layer. Final upper layer was separated and 0.7 volume of chilled isopropanol was added. Tubes were kept at -20 °C for 1 h. DNA was then pelleted by centrifuging at 15,000 rpm 10 min at 4 °C. This was followed by washing of the pellet with 1.0 mL of 70% ethanol. Finally, pellet was air-dried and dissolved in 50 µl of 1X TE. Method III (Physiochemical) This method is based on the protocol of Bag et al. [9]. Fecal material 1.0 g was homogenized in 1.0 mL of 1X PBS using vortex in tube containing sterile 5 glass beads (3-5 mm). Tubes were allowed to stand for 10 min and then supernatant was transferred to a new tube. To this 100 µl of lysozyme (20 mg/mL) was added and incubated overnight at 37 0 C. Then 500 µl of guanidine thiocyanate (4.0 M) was added and mixed gently. This was followed by the addition of 600 µl of 10% N-lauryl sarcosine and 40 µl of Proteinase K (20 mg/mL). Mixture was incubated at 65 °C for 2 h. Then 200 mg of sterile zirconium beads were added and vortexed at high speed for 5 min. Mixture was then centrifuged at 5000 rpm for 5 min. Supernatant was collected in a fresh tube. To this 400 µl of 5.0 M NaCl and 320 µl of CTAB was added and incubated at 65 °C for 30 min. Then an equal volume of chloroform-isoamyl alcohol (24:1) was added and centrifuged at 10,000 rpm for 15 min at 4 °C. The step was repeated using the upper layer.
To the final upper layer, 1/10 volume of 3.0 M sodium acetate was added followed by addition of a double volume of chilled absolute ethanol. Tube was left overnight in -20 °C followed by centrifugation at 4 0 C for 10 min at 15,000 rpm. Pellet was washed with 1.0 mL of 70% ethanol. Final pellet was dried and dissolved in 50 µl of PCR grade water. Method IV (Chemical) This method is based on the Qiagen Stool Lysis Buffer. Fecal material 1.0 g was homogenized in 5.0 mL lysis buffer by vortexing for 5 min. Mixture was incubated at 70 0 C for 10 min and cooled to RT. Then centrifuged at 8000 rpm for 1 min. Supernatant was used as template in PCR. Method V (physical) This is a previously described method of Stabel et al. [10], with few modifications. Feces (1.0 g) were homogenized in 9.0 mL of IX TE using motor pestle. Mixture was transferred to 15 mL tubes and vortexed for few seconds. Mixture was allowed to settle followed by vortexing again for few seconds. Tubes were centrifuged at 200 g for 30 s. Resulting supernatant was transferred to a fresh tube. Supernatant was diluted to 1:100 using 1X TE. Then 1.0 mL of the diluted sample was transferred into a tube. Sample was centrifuged at 13,000 g for 2 min. Supernatant was discarded, and pellet was washed twice with 1.0 mL of 1X TE followed by centrifugation for 2 min at 13,000 g. Final pellet was suspended in 200 µl of 1X TE. These tubes were placed in boiling water 10 min. Mixture was cooled to RT and RNAase A was added at concentration of 1.0 µg/ mL. This was used as template in fecal PCR.

IS900 PCR
DNA amplification was done utilizing MAP specific IS900 primers (FP: 5'-CCG CTA ATT GAG AGA TGC GAT TGG − 3'; RP: 5'-AAT CAA CTC CAG CAG CGC GGC CTC G-3'). Study evaluated ready to use PCR master mix (PRM) from three sources (Qiagen, Genei and Thermo) with additional PCR additives in nine different combinations (Table 1). Initially, all PRMs were used to test fecal DNA isolated from spiked fecal samples using five DNA extraction methods. Combination of DNA isolation method and PRM giving best sensitivity on spiked was used to test reference and field fecal samples. Positive and negative control was also run along with test samples. Thermal cycling conditions included: 5 min of initial denaturation at 94 °C, followed by 40 cycles of denaturation at 94 °C for 30s, followed by annealing at 64 °C for 30s, extension at 72 °C for 30s, and the final extension at 72 °C for 10 min. Amplification product of 229 bp in 1.5% agarose gel revealed positive reaction.

µl
Forward primer Reverse primer MgCl 2− Thermo fisher (25 mM) MgCl 2 -Thermo fisher (25 mM) 3.5 µl Genomic DNA 2.0 µl PRM: PCR Master Mix helps in getting rid of primer dimers and reduces the melting temperature of template. There are certain novel DNA sequences that cause pause of DNA polymerase. Presence of betaine helps polymerase to avoid these pauses [12][13][14]. BSA is particularly helpful in stabilizing the Taq polymerase; helps in amplification of old DNA and getting rid of inhibitors [15,16].
On the reference fecal samples sensitivity was 83% and some of samples were missed in PCR using this combination of DNA isolation and PRM. This can be attributed to low initial load of MAP in these samples due to longer durations of storage over the years in freezing conditions that may have resulted in damage to MAP cells. High positivity of field animals is attributed to the fact that these animals belonged to herds/farms with history of clinical incidences of paratuberculosis. Previous workers have also reported a high prevalence of paratuberculosis in the country [17][18][19][20]. This study is in agreement with previous studies wherein cattle were the most susceptible species for paratuberculosis [20,21]. These findings reveal that cattle are more susceptible to paratuberculosis compared to other species. Crossbreeding with semen from imported bulls may be a reason for the low immunity of our cattle.

Conclusion
It can be concluded from the findings of the present study physical fecal DNA isolation in combination with Genei based master mix DNA is best suited protocol for in-house fecal IS900 PCR to diagnose paratuberculosis especially in the view of resource limited areas. Further, high prevalence of paratuberculosis needs immediate attention and implementation of the control programs.

Results and discussion
On the spiked fecal samples, DNA isolation method V with PRM IV gave best results in terms of limit of detection (10 2 CFU/g) ( Table 2). Hence, this combination was selected for further testing. Of the 100-reference positive fecal samples 83 were positive using this combination of DNA isolation method (V) and PRM (IV). None of the reference negative samples appeared positive. Sensitivity and specificity of this combination was 83% and 100%, respectively. Field fecal samples (n = 521) were tested using Method V of fecal DNA extraction and PRM IV. Cumulatively, of the 521 samples, 120 (23.03%) were positive. Species wise highest positivity was observed in cattle (32.8%) followed by goat (20%), buffalo (19.3%) and sheep (11.3%).
Findings revealed that the physical method of DNA isolation (Method V) showed the highest diagnostic sensitivity when used with PRM IV. Previously, Stable et al., [10] have also found this method of DNA isolation highly sensitive and reproducible. Similarly, Haghkhah et al. [11] also reported that this DNA isolation method is useful in IS900 PCR. Recovery of DNA in this method is based on the concentration of cells followed by lysis using heating, therefore, chances of losing DNA are highly unlikely compared to other multi-step methods and there is a risk of losing DNA at each step. Genei based ready master mix with additives was better over Qiagen and Thermo. Reason for the success of the Genei based master mix cannot be figured out because the exact compositions of these master mixes are unknown. It was observed that increasing MgCl 2 concentration decreased the performance of PCR (Tables 1 and  2). Higher concentrations of Mg 2+ , interferes with denaturation of the DNA template by stabilizing the duplex strand [12]. Further, higher concentrations of Mg 2+ can stabilize annealing of primers to incorrect template sites and thereby decreases the overall performance of PCR [2]. Other PCR additives included betaine, DMSO and BSA. Betaine and DMSO helps in reducing the secondary structure in templates during denaturation and amplification process, thereby enhances amplification [12][13][14]. Further, DMSO  I PRM II PRM III PRM IV PRM V PRM VI PRM VII PRM VIII PRM IX  10 6 -