Optimized PCR and RPA assay
The primer pair RPA11F/RPA11R was found to be efficient in amplifying the 126 bp LipL32
gene from twenty-three pathogenic reference strains and not from the non-pathogenic Leptospira by PCR. The PCR assay targeting the pathogenic gene is considered a confirmative diagnosis (OIE, 2021a)
. Hence lipL32
gene was chosen as the target for both the PCR and the RPA assay, which is highly expressed during infection (Haake et al., 2000).
For the RPA assay, 24 pmol of each of the primers, 2.8 mM Magnesium acetate (0.5 µl), 20 ng of template DNA, incubation temperature of 39°C and a reaction time of 30 minutes were found to be the optimized conditions for amplification of 126 bp amplicon targeting LipL32
gene (Fig 1) of pathogenic leptospira. The RPA assay efficiently amplified the lipL32
gene (126 bp) from the reference strain of pathogenic serogroups. It is in concordance with the application of RPA assay to detect the methicillin-resistant Staphylococcus aureus (Piepenburg et al., 2006), Leptospira
sp (Ahamed et al., 2014), Mycobacteriu
m sp (Singpan omchai et al., 2019).
Analytical performance of the developed RPA assay
During ascertaining the variability in pipetting of the reaction components, the coefficient of variation ranged from 0.3 to 1.1% was noticed, indicating negligible contribution for dispensing the respected volumes with the micropipettes. The concentration of the stock DNA was 53 ng/ìl which is equivalent to 1.014×107 copies. On serial dilution and detection of genomic copies, the lower limit of detection for developed RPA assay and PCR assay was estimated as 102 copies. The RPA assay amplified the LipL32
gene from pathogenic reference strains but not from non-pathogenic Leptospira and other Gram-negative bacterial species such as Brucella, Salmonella
DNA, confirming the specificity of the RPA assay. This sensitivity and specificity in detecting Leptospira DNA confirm the analytical characteristics of the optimized RPA assay.
Diagnostic characteristics of the developed RPA assay
The PCR assay detected 67 samples as positive, while 64 samples were found to be positive by RPA assay. The test positivity to Leptospira
DNA in the samples was 42.6% (64/150) and 44.6% (67/150) by the RPA assay and PCR respectively. The test results indicated a good diagnostic agreement between RPA assay and PCR with a kappa value of 0.905 (0.837 to 0.974 at 95% CI). The diagnostic sensitivity and specificity of RPA assay to detect Leptospira
DNA when applied to clinical samples was 92.5% (83.44% to 97.53%, 95% CI) and 97.59% (91.57% to 99.71%, 95% CI) respectively, in comparison with PCR (Table 2).
Table 2: Performance of RPA assay in comparison with the PCR assay for detecting leptospira DNA in clinical samples.
However, the RPA assay detected leptospiral DNA from two samples that tested negative by the PCR assay, the failure in the PCR assay could be due to the presence of inhibitory factors in the sample (Ahamed et al., 2009).
When applied to the kidney tissues and water samples, both RPA assay and PCR detected Leptospira
DNA in twelve tissue and ten water samples showed a similar positivity percentage. It implies the use of the assay for surveillance of Leptospira in the environment which is considered to be a source of infection.
The real-time PCR detected Leptospira DNA in 74 samples with a positive rate of 49.3% (74/150). None of the samples that tested negative on either PCR or RPA assay was found to be positive on real-time PCR assay confirming the diagnostic specificity but showed slightly higher sensitivity than both the assays. The high sensitivity in real-time PCR assay is due to the inherent ability of the fluorescent tags that are used as the reporter (Thaipadunpanit et al., 2011).
Repeatability and reproducibility characteristics of the developed RPA assay
The mean DNA concentration of amplified products on different occasions was 42.24 ng/ul ± 1.44 with a coefficient of variation of 5.9% indicating good repeatability of the assay. Out of 25 blind-coded samples that were used for the reproducibility assessment, the Leptospira DNA was detected in 19 samples in the third-party testing laboratory, while 17 samples tested positive in this laboratory. Analysis of the results revealed better agreement on the performance of the assay across laboratories with a kappa value of 0.81 (0.547 to 1.00 at 95% CI ). The reported RPA assay showed very good intra and inter-assay repeatability and reproducibility. The repeatability of the assay performed on different occasions showed a co-efficient of variation of 5.9% is minimal and in agreement with the report of Reed et al., (2002).
The removal of DNA binding protein by heat as an alternative method in the study, before visualization by gel electrophoresis, reduces the time and expenses, compared to the manufacturer’s recommended method.
RPA assay extends the capabilities of diagnostic facilities without access to a thermocycler and generates the result in 30 minutes, but the reagent is costly. On factoring, in the time and cost of equipment, RPA assay is less expensive than PCR assay. Taken all together, the RPA assay is a promising tool for canine leptospirosis detection, which is simple, rapid and reliable in resource-limited diagnostic laboratories and on-site facilities. Further, simplification is possible by using the endpoint detection through the lateral flow platform as a point care test, during an outbreak for early diagnosis of canine leptospirosis.