Brucellosis - a zoonotic disease has been attracting much attention in recent times in both human as well as veterinary medicine
(Ohtsuki et al., 2008). Since there are no pathognomonic signs and symptoms in man and animals suffering from brucellosis, diagnosis relies on serological and molecular tests. Though a number of serological tests are available, each has its own shortcomings. In order to overcome these drawbacks and for high sensitivity and specificity, many researchers have used molecular diagnostic tests
(Spicic et al., 2010; Asaad and Algahtani, 2012;
Al Dahouk et al., 2013). These tests require trained man power and sophisticated equipments. Thus, there is a need for simple, rapid, sensitive, specific and easy-to-perform test for fast and accurate diagnosis of brucellosis in human and in animals.
In this regard, the LAMP assay has many advantages as it is rapid, simple and easy to perform. Unlike other molecular tests, it does not need any sophisticated equipment rather requires a water-bath and can be performed in peripheral laboratories and the results can be read with naked eyes.
The DNA hybridization studies have suggested that the
Brucella species (
B. abortus, B. melitensis, B. suis, B. ovis, B. canis and
B. neotomae) represent a monospecific genus or a genomic species
(Verger et al., 1985). Brucellae encode two major outer membrane proteins of 25kDa and 36kDa. The former one is encoded by
omp25 gene whereas the later was encoded by two closely related genes
i.e. omp2a and
omp2b (
Marquish and Ficht, 1993;
Cloeckaert et al., 1995); Omp2a porin shows size variation and in certain
B. abortus biovars
i.e. biovars 1, 2 and 4, a large deletion of approx. 30 residues is reported
(Mobasheri et al., 1997). The importance of these major outer membrane protein genes were clearly emphasized for diagnostic purpose
(Cloeckaert et al., 1995).
The present close-tube LAMP based detection strategy of
Brucella genome was found simple and easy to carry out. In the present study, the SYBR Green dye was put underneath the cap of PCR tube. Due to surface tension, the SYBR Green drop usually sticks to the underneath of the cap of PCR tube until the reaction has come to an end. Hence, there was no need to open the cap of PCR tube on completion of amplification. Thus, the major source of persistent contamination was avoided. Moreover, the vaporization of reaction mixture and coming in contact to the SYBR Green was checked by adding a layer of sterilized liquid paraffin above the reaction mixture. Post-amplification, spin of few seconds resulted in coming down of the SYBR Green dye from the cap to the amplified mixture passing through the liquid paraffin layer due to its specific gravity. A volume of 1.5μl of SYBR Green was found better than using 1μl during detection, as there might be loss of certain fraction of dye due to sticking on the cap and walls of the tube.
There are several closed-tube formats reported earlier
i.e. tin foil method
(Hong et al., 2012), wax bullet method
(Tao et al., 2011; Liang et al., 2013) and agar-dye capsule method
(Karthik et al., 2014). However, it is easy to perform than earlier formats. It is difficult to prepare wax bullets containing fluorescent dye in resource limited laboratories and field conditions. Similarly, agar capsules have to be prepared every time fresh before reaction mixture and these cannot be stored at deep fridge temperature (-20°C), which is essential for efficacy of SYBR Green dye incorporated into the capsule. Furthermore, the detection in visible light may be interfered or misinterpreted due to presence of agar inside the amplification mixture, which may be confused with the white precipitates
i.e. magnesium pyrophosphate. Moreover, there is no need of boiling at the end of amplification unlike wax bullet/capsule methods. Reaction can be carried out in ordinary water bath and there is no need of any special equipment like PCR thermal cycler and commercially available tube scanners. In earlier reports, placement of microcrystalline wax capsule and agar dye capsules are needed in such a way that it (wax and agar respectively) should not come in contact to the reaction mixture, unlike the present version where sterile liquid paraffin is simply overlaid on the reaction mixture before amplification, similar to the report described earlier
(Zhang et al., 2013).
In brief, the major advantages of the reported SYBR Green drop format are as follows. There was incorporation of the dye in its actual form without any special modification; secondly, adoption of closed-tube format that swap over the contamination problem and thirdly, performance of the technique in simple water bath suitable for resource limited laboratories and having actual field applicability.
In the present report, amplification was marked in tubes through naked eye by observing white precipitates after completion of the amplification. Yellowish green fluorescence was also observed by adding 1.5μl SYBR Green 1 dye (1000X) to the amplified product. Amplification was found to be optimal at 63°C. Amplification was observed in tubes after 60 min,
i.e., after 60, 70 and 80 min but not after 20 or 40 min (Fig 2). The reaction condition was standardized at 63°C for 70 min for the amplification using both set of primers without incorporation of loop primers.
Subsequently, the amplification condition was further optimized using loop primers to detect significant change in duration of amplification. Optimization was carried out using second set of primers as it was found more sensitive than first set of primers. On optimization of the standardized
omp2b gene based LAMP assay, it was found that the fluorescence was observed in 55 min using both the loop primers (forward and reverse), while fluorescence was observed in 40 min using single loop primer (forward only).
Moreover, while testing for false amplification by subjecting the no-template control tubes for 2 hrs and 2.5 hrs, there was no white precipitates formed in any of the tubes but fluorescence was observed after adding SYBR Green I dye in both of the no-template controls, indicating the false amplification that was resulted due to increase in duration of amplification. Test for false amplification was carried out to know the significance of duration of amplification.
Analytical specificity and relative sensitivity
The analytical specificity of LAMP was demonstrated by absence of amplification in any of the non-
Brucella species against presence of white precipitation and greenish fluorescence in all of the
Brucella species (Table 3). The relative sensitivity was compared with
bcsp31 PCR, reported to be one of the most sensitive genus specific PCR for detection of
Brucella (
Yu and Nielsen, 2010). The present LAMP assays was found to be more sensitive as it could detect 0.34pg of genome using first set of primers while 34fg of genome using second set of primers (Fig 3) against 3.4pg by
bcsp31 PCR (Fig 4). It was inferred from the result that the second set of primers were found much suitable as compared to first as it was found to be 100 times more sensitive as compared to
bcsp31 PCR. Similarly, in earlier reports, the detection limit was found to be 10fg for first set of primers and 0.1pg for second set of primers for
bcsp31-LAMP
(Ohtsuki et al., 2008). Further,
omp25-LAMP was reported to be 10 times more sensitive than PCR
(Song et al., 2012; Karthik et al., 2014). IS711-LAMP was found 100 times more sensitive than conventional PCR
(Zadon et al., 2014). However,
omp2b gene based LAMP assay has taken in to consideration more divergent group of
Brucellae i.e. five most common classical
Brucella species
viz.
B. abortus,
B. melitensis,
B. suis,
B. canis and
B. ovis.
Restriction digestion analysis
On restriction digestion of the amplified product of first set of primers with
HaeIII, 3 bands at 43bp, 160bp and 203bp were observed in agarose gel electrophoresis (Fig 5). Similarly, the amplified products of second set of primers resulted in 216bp and 226bp fragments with
HpaII digestion and 3 bands of 49bp, 179bp and 221bp with
HhaI digestion (Fig 6). The restriction digestion yielded definite bands confirming that the technique developed was specific for
Brucella DNA. Similarly, specific restriction patterns were obtained in
bcsp31-LAMP with
Sau3AI digestion (135bp and 241bp) and
EcoRV digestion (176bp and 250bp)
(Ohtsuki et al., 2008); IS711-LAMP with
RsaI digestion (75bp, 105bp and 113bp)
(Perez-Sancho et al., 2013). The restriction patterns were expected as per the method described earlier
(Notomi et al., 2000).
Feasibility of omp2b gene based LAMP assay for clinical samples
Many of the experimentally infected as well as clinical samples were assessed through various LAMP techniques in earlier studies. However, milk was found to be most popular and convenient sample for disease diagnosis in field condition, although intermittent excretion of pathogens was a matter of consideration. On assessment of milk sample,
omp2b gene based LAMP detected 4.75 × 10
1 CFU/ml of Brucellae in spiked milk samples. However,
bcsp31-LAMP was able to detect 4.9 × 104 CFU/ml of pathogen in
Brucella contaminated milk
(Ohtsuki et al., 2008). However, detection limit was found much less in spiked semen sample (4.75 × 10
7 CFU/ml) in comparison to milk in the present study. The inconsistent result might be due to its nature of sample. Similar data are available pertaining to detection limit in various other diverse samples by many earlier studies
i.e. 2.2 × 10
3 CFU/ml (infected mice liver), 8.2 × 10
2 CFU/ml (infected mice spleen)
etc.
(Ohtsuki et al., 2008).
