Application of a Multiplex PCR Assay for Molecular Identification of Pathogenic and Non-Pathogenic Leptospires based on lipL32 and 16S rRNA Genes

1. Introduction

Leptospirosis is an acute febrile illness caused by pathogenic Leptospires. This zoonotic disease has a worldwide distribution but is most common in tropical and subtropical regions and has the greatest impact on public health in developing countries ( 1 , 2 ). The infection of humans occurs by direct or indirect contact with the urine of infected animal ( 3 , 4 ).

Laboratory diagnosis of leptospirosis is based on several methods such as culture, serology methods like MAT and ELISA, molecular detection (PCR) ( 5 - 7 ). Culture has low sensitivity and requires specialized expertise and is time consuming ( 6 , 8 ). Although MAT is used as a reference method for diagnosis in various studies, it is not a suitable method because it is based on the use of a live bacteria, with false negative results (low sensitivity) in the early course of infection and is time consuming ( 9 , 10 ).

PCR has been used to detect leptospiral DNA in samples obtained from animals ( 11 , 12 ) and human ( 13 , 14 ), with quick results and high sensitivity. This method is superior to the methods of culture and MAT ( 13 , 14 ). The multiplex PCR method is used for rapid and simultaneous detection. This method allowed detection of several PCR products ( 3 ).

The lipL32 gene is a major outer-membrane lipoprotein from the genus Leptospira. ( 15 ) LipL32 is highly conserved among pathogenic Leptospira species ( 15 , 16 ) but has no present in the saprophyte Leptospira biflexa ( 17 ). The 16S rRNA gene is used for phylogenetic studies ( 18 ) because it is highly conserved between different species of bacteria ( 19 ). However, 16S rRNA gene sequence has hypervariable regions, which can be used to identify bacteria. Identification of pathogenic and non-pathogenic leptospirea has been used by 16S rRNA gene ( 20 , 21 ).

Therefore, the purpose of this study was molecular detection of pathogenic and non-pathogenic Leptospira by Multiplex PCR assay based on lipL32 and 16Sr RNA genes.

2. Materials and Methods

2. 1. Bacterial Serovars

The bacterial serovars used in the study are presented in table 1. All serovars obtained from the Leptospira Reference Laboratory, Department of Microbiology, Razi Vaccine and Serum Research Institute, Karaj, Iran.

2. 2. Culture

The bacteria were subcultured in a selective culture medium of EMJH with rabbit serum and enrichment supplements under aerobic conditions at 28 ° C for 7 - 10 days. Their growth was investigated by a dark field microscope.

2. 3. DNA Extraction

Culture isolate were centrifuged at 17000 X g for 20 min at 4 ° C. The genomic DNA was extracted by the standard phenol-chloroform method. The quality and quantity of the extracted DNAs were evaluated by electrophoresis and by UV spectrophotometry and analyzed by 1% agarose gel electrophoresis. DNA was stored at -20°C until used for PCR.

2. 4. Multiplex PCR

In this study, we have used the Multiplex PCR method with 2 sets of primers, which will aid in the specific and sensitive detection of Leptospira species.

The characteristics of primers used in this study are presented in table 2.

The LipL32 PCR assay was done to amplify a 272bp fragment from pathogenic Leptospira. The Lipl32 gene is specifically pathogenic leptospiral and is present only in pathogenic serovars.

The 16S rRNA PCR assay was done to amplify a 240bp fragment from saprophytic Leptospira, using previously described specific primers ( 20 ). These primers were expected to amplify saprophytic leptospiral DNA.

These two sets of primers allowed us to distinguish between pathogenic and saprophytic Leptospira species. Lipl32 gene is amplified in pathogenic species and 16S rRNA is amplified in the saprophytic species.

To determine the proper concentration of each primer, the values of 5, 10, 15 and 20 pmol of primers were evaluated in the PCR test. Sensitivity testing was performed for both pairs of primers. The sensitivity of the PCR was determined using a serial dilution of genomic DNA leptospiral.

Multiplex PCR amplification was performed in a final reaction volume of 16 μL. It consisted of 8 μl 2X Master Mix, 1 μl (10 pM) from each of the forward and reverse primer and 10 ng of template DNA. The PCR was performed in thermocycler (Eppendorf, Germany). The reaction mixture was initiated by incubation at 95 ºC for 5 minutes, followed by 35 cycles of denaturation at 95 ºC for 30 seconds, annealing at 61 ºC for 30 seconds, extension at 72 ºC for 1 minute, and a final extension at 72 ºC for 10 minutes.

The PCR products were electrophoresed on 1% agarose gels and stained with safe staining (Sinagen). Gels were photographed using a gel documentation system (Bio-Rad Chemi XRS Gel Documentation system, UAS).

3. Results

Initially, the PCR was performed separately with each primer. The PCR products of pathogenic Leptospira amplified by lipl32 primer visible bands with molecular size of 272bp. The bond was not observation in non-pathogenic Leptospira and Salmonella enteritidis (as negative control). For detection of saprophytic Leptospira, 16S rRNA gene were used with molecular size 240bp. This fragment was not detected in pathogenic Leptospira and Salmonella enteritidis. The sensitivity for lipl32 and 16S rRNA primers were 10^-4 pg / μl and 10^-6 pg / μl respectively. The sensitivity for multiplex PCR was 10^-3pg / μl.

3. 1. Multiplex PCR

In multiplex PCR, were used two pairs of primers simultaneously. The PCR products of pathogenic Leptospira amplified by lipl32 primer (272bp) whereas non-pathogenic Leptospira showed the 240 bp of 16S rRNA only (Figure 1).

Figure 1. Agarose gel electrophoresis of multiplex-PCR products showing the detection of pathogenic and non-pathogenic leptospires. Lane 1:100 bp DNA ladder; Lan 2: non-pathogenic Leptospira (L.biflexa Serovar Semaranga RTCC 2819); Lan 3: non-pathogenic Leptospira (L.biflexa Serovar Semaranga RTCC 2828); Lane 4: pathogenic Leptospira (L.interrogans Serovar Canicola RTCC 2805); Lan 5: pathogenic Leptospira (L.interrogans Serovar Grippotyphosa RTCC 2808); Lan 6: Salmonella enteritidis (RTCC 1621)

In this Multiplex PCR reaction, pathogenic and non-pathogenic serovars were separated from each other.

According to the figure 1, The PCR products of pathogenic and non-pathogenic Leptospira by lipl32 and 16S rRNA primers, were respectively produced visible bands with molecular size of 272 bp and 240 bp. In this way, pathogenic and non-pathogenic serovars were separated. The Figure lanes 2, 3 shows the positive amplification of only the 16S rRNA gene for non-pathogenic Leptospira (240bp) and lanes 4, 5 shows the positive amplification of only the lipL32 gene for pathogenic Leptospira (272bp).

These two sets of primers allowed us to distinguish between pathogenic and saprophytic Leptospira species. Both genes are amplified in Leptospira genus (lipL32 in pathogenic Leptospira and 16S rRNA in non-pathogenic Leptospira).

4. Discussion

Leptospirosis is one of the most common zoonoses in the world. Non-pathogenic and pathogenic leptospires are morphologically similar to each other and culture is very slow and time consuming method. PCR is used to identify a large number of microorganisms, including important clinical microorganisms. The PCR reaction has a high sensitivity and no need culture microorganisms. As a result, PCR is a good way to identify the organisms involved in acute infections ( 2 ).

The advantage of PCR for detecting leptospirosis is due to its ability to detect Leptospira in the early stages of the disease. Leptospirosis is an acute illness and spreads rapidly. Therefore, an appropriate laboratory test such as PCR with high sensitivity and specificity is required for diagnosis ( 22 ).

The purpose of this study was to evaluate Multiplex PCR method for identifying pathogenic and non-pathogenic Leptospira simultaneously using two pairs of primers. The Multiplex PCR used in this study offers further advantage over that of conventional PCR because it uses 2 sets of primers that increase the specific amplification (272 bp and/or 240 bp).

The targeting of the two genes in the Multiplex PCR allows us to more precisely reach the final diagnosis. In this study, using multiplex PCR method and using two pairs of specially designed Lipl32 primers of pathogenic Leptospira and specific 16S rRNA of non-pathogenic Leptospira, these two species of Leptospira were found to be much easier than conventional methods.

Tansuphasiri, Thipsuk ( 3 ) in 2006 for detected pathogenic serovars and genus Leptospira used LipL32 primers with lengths of 279 bp and 16S rRNA with 430 bp length. The sensitivity of primers in this experiment was 1pg. In this study, two pairs primers (LipL32 and 16S rRNA) were identified as pathogenic and non-pathogenic serovars. The sensitivity of the primers LipL32 and 16S rRNA was 10 ^-4 pg and 10 ^-6 pg respectively. The sensitivity of our research was more than research Tansuphasiri et al. In addition, the 16S rRNA primer in the Tansuphasiri study could not detect non-pathogenic serovars ( 3 ).

In 2006, Leon, Pronost ( 23 ) performed a multiplex PCR test using three primer pairs of hap1 (262bp), 23S rRNA (248bp) and 16S rRNA (240bp). The hap1 primer was able to detect pathogenic serovars. 16S rRNA Primer detected non-pathogenic serovars and the 23S rRNA primer was effective in identifying the genus Leptospira ( 23 ).

The distinction between the findings of this study and Leon's studies was that we used LipL32 to detect pathogenic serovars, but Leon used hap1. Subscription these two studies were based on the use of the 16S rRNA primer that was able to detect non-pathogenic serovars. The results of the 16S rRNA primers are common in both studies.

Moreno and Agudelo-Florez ( 24 ) in 2010 to implement and validate conventional and multiplex PCR methods using lipL32 and secY/flaB genes to assess the capacity of PCR methods to identify pathogenic and saprophytic species of Leptospira ssp. In this study 22 international reference strains and 12 colombian isolates were used. Best results were obtained with the lipL32 PCR, which displayed a better sensitivity and a better capacity to detect different strains than the multiplex PCR. The secY primers showed a poor specificity to pathogenic species and a poor sensitivity. Thus, lipL32 primers show high potential for molecular diagnosis of Leptospira spp in clinical and environmental samples ( 24 ).

Ahmed, Sandai ( 15 ) in 2012 developed a multiplex PCR assay for detecting Leptospira’s DNA. The multiplex PCR assay detected both the LipL32 primers with lengths of 330 bp and 16S rRNA with 660 bp length. In this method from 10 species of Leptospira and 23 other species of bacteria were used. A positive result was obtained from all leptospiral serovars. In this study, pathogenic and non-pathogenic serovars were not distinguished from each other, but in the present study pathogenic and non-pathogenic serovars were identified from each other ( 15 ).

Our results suggested that that multiplex PCR can be used to detect Leptospira samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much easily than conventional methods such as culture and MAT.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

1. Ko AI, Goarant C, Picardeau M. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009; 7(10):736-47.
2. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001; 14(2):296-326.
3. Tansuphasiri U, Thipsuk C, Phulsuksombati D, Chanyasanha C. Duplex PCR-hybridization based detection of pathogenic Leptospira in environmental water samples obtained from endemic areas in northeast region of Thailand. Southeast Asian J Trop Med Public Health. 2006; 37(4):729-41.
4. Vijayachari P, Sugunan AP, Shriram AN. Leptospirosis: an emerging global public health problem. J Biosci. 2008; 33(4):557-69.
5. Fahimipour A, Khaki P, Moradi Bidhendi S. Seroepidemiological Analysis of Leptospiral infection using MAT in Stray Dogs in Alborz, Iran. Arch Razi Inst. 2021; 76(2):391-6.
6. Khaki P. Clinical Laboratory Diagnosis of Human Leptospirosis. Int J Enteric Pathog. 2016; 4(1):1-31859.
7. Musso D, La Scola B. Laboratory diagnosis of leptospirosis: a challenge. J Microbiol Immunol Infect. 2013; 46(4):245-52.
8. Levett PN, Morey RE, Galloway RL, Turner DE, Steigerwalt AG, Mayer LW. Detection of pathogenic leptospires by real-time quantitative PCR. J Med Microbiol. 2005; 54(Pt 1):45-9.
9. Cumberland P, Everard CO, Levett PN. Assessment of the efficacy of an IgM-elisa and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis. Am J Trop Med Hyg. 1999; 61(5):731-4.
10. Niloofa R, Fernando N, de Silva NL, Karunanayake L, Wickramasinghe H, Dikmadugoda N, et al. Diagnosis of Leptospirosis: Comparison between Microscopic Agglutination Test, IgM-ELISA and IgM Rapid Immunochromatography Test. PLoS One. 2015; 10(6):e0129236.
11. Bomfim MR, Barbosa-Stancioli EF, Koury MC. Detection of pathogenic leptospires in urine from naturally infected cattle by nested PCR. Vet J. 2008; 178(2):251-6.
12. Lilenbaum W, Varges R, Brandao FZ, Cortez A, de Souza SO, Brandao PE, et al. Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction. Theriogenology. 2008; 69(7):837-42.
13. Merien F, Amouriaux P, Perolat P, Baranton G, Saint Girons I. Polymerase chain reaction for detection of Leptospira spp. in clinical samples. J Clin Microbiol. 1992; 30(9):2219-24.
14. Ooteman MC, Vago AR, Koury MC. Evaluation of MAT, IgM ELISA and PCR methods for the diagnosis of human leptospirosis. J Microbiol Methods. 2006; 65(2):247-57.
15. Ahmed SA, Sandai DA, Musa S, Hoe CH, Riadzi M, Lau KL, et al. Rapid diagnosis of leptospirosis by multiplex PCR. Malays J Med Sci. 2012; 19(3):9-16.
16. Khodaverdi Darian E, Forghanifard MM, Moradi Bidhendi S, Chang YF, Yahaghi E, Esmaelizad M, et al. Cloning and Sequence Analysis of LipL32, a Surface-Exposed Lipoprotein of Pathogenic Leptospira Spp. Iran Red Crescent Med J. 2013; 15(11):e8793.
17. Haake DA, Chao G, Zuerner RL, Barnett JK, Barnett D, Mazel M, et al. The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun. 2000; 68(4):2276-85.
18. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991; 173(2):697-703.
19. Coenye T, Vandamme P. Intragenomic heterogeneity between multiple 16S ribosomal RNA operons in sequenced bacterial genomes. FEMS Microbiol Lett. 2003; 228(1):45-9.
20. Murgia R, Riquelme N, Baranton G, Cinco M. Oligonucleotides specific for pathogenic and saprophytic leptospira occurring in water. FEMS Microbiol Lett. 1997; 148(1):27-34.
21. Pereira F, Carneiro J, Matthiesen R, van Asch B, Pinto N, Gusmao L, et al. Identification of species by multiplex analysis of variable-length sequences. Nucleic Acids Res. 2010; 38(22):203.
22. Mulla S, Chakraborty T, Patel M, Pandya HP, Dadhaniya V, Vaghela G. Diagnosis of leptospirosis and comparison of ELISA and MAT techniques. Indian J Pathol Microbiol. 2006; 49(3):468-70.
23. Leon A, Pronost S, Tapprest J, Foucher N, Blanchard B, Andre-Fontaine G, et al. Identification of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain reaction analysis. J Vet Diagn Invest. 2006; 18(2):218-21.
24. Moreno N, Agudelo-Florez P. [Application of conventional and multiplex PCR assays for identification of isolates of Leptospira spp. in Colombia]. Rev Peru Med Exp Salud Publica. 2010; 27(4):548-56.