WO2012066576A2

WO2012066576A2 - Oligonucleotide primer sequences for detection of leptospira

Info

Publication number: WO2012066576A2
Application number: PCT/IN2011/000797
Authority: WO
Inventors: Martin Annette; Kashinath Khonde Vinaykumar; Subhash Shukre Kedar
Original assignee: Godavari Biorefineries Limited
Priority date: 2010-11-16
Filing date: 2011-11-16
Publication date: 2012-05-24
Also published as: WO2012066576A3

Abstract

The present invention is directed towards providing oligonucleotide primer sequences for the detection of Leptospira species in biological and environmental samples. The oligonucleotide primer sequences of the present invention are capable of detecting DNA of Leptospira species in a given sample using molecular protocol for example a polymerase chain reaction (PCR) technique. The present invention also provides a method and a kit for detecting leptospiral DNA in a given sample with high specificity.

Description

TITLE

OLIGONUCLEOTIDE PRIMER SEQUENCES FOR DETECTION OF LEPTOSPIRA. FIELD OF THE INVENTION

The present invention relates to oligonucleotide primer sequences for detection of Leptospira species in biological and environmental samples. More particularly the present invention relates to oligonucleotide primer sequences for detection of Leptospira species using molecular protocol.

BACKGROUND OF THE INVETION

Leptospira is a genus of spirochaete bacteria, including a small number of pathogenic and saprophytic species. Members of Leptospira are also grouped into serovars according to their antigenic relatedness. There are about 20 species of Leptospira and currently over 200 recognized serovars. Spirochetes of the genus Leptospira infect animals and humans and are the causative agents for the emerging infectious disease leptospirosis that affect humans and a wide range of animals, including mammals, birds, amphibians, and reptiles.

The bacterial spirochete colonizes the renal tubules of infected animals and is shed in the urine for varying periods of time, depending on whether the animal is an accidental or reservoir host for that serovar. Rats are reservoir hosts for serovars of Leptospira interrogans belonging to the lcterohaemorrhagiae serogroup, and infected rats probably remain infected, and infectious, for life. The infection is commonly transmitted to humans by allowing water that has been contaminated by animal urine to come in contact with unhealed breaks in the skin, the eyes, or with the rnucous membranes. In tropical regions of the world, leptospirosis is a widespread public health problem. The disease is endemic wherever open sewers or agricultural practices lead to contamination of water with animal urine. Although most human leptospirosis infections are self-limited, complications are common, involving hepatorenal failure, pulmonary hemorrhage, and death in 10%-50% of severe cases. W.H.O report states that the prevalence in tropical countries is nearly 100 per 100000 populations.

Highest prevalence of leptospirosis is after the rainy season when heavy rainfall and inadequate civil engineering result in urban flooding. Given the laborious nature of leptospiral isolation and culture techniques, particularly from environmental samples, there was no systematic method available to determine the identity, spectrum, and density of leptospires in water samples. Problem with the detection of leptospirosis in clinical samples is that due to the wide range of symptoms the infection is often wrongly diagnosed. Other challenges in detecting Leptospirosis on clinical samples include the similarity of signs and symptoms with those of other bacterial, viral and parasitic infections and frequent occurrence of the disease in atypical forms.

The MAT (microscopic agglutination test), is considered the gold standard in diagnosing leptospirosis, however a large panel of different leptospira needs to be subcultured frequently, which is both laborious and expensive due to which this method is underused. The need for paired serum samples and delayed antibody response do not contribute much to the early diagnosis. Moreover, when the causative Leptospira serovar is not included in the panel of typing organisms, it frequently results in false negative results. Several other alternatives of antibody detection assays have subsequently been developed for diagnosis of leptospirosis. These include the hemolytic test, the slide agglutination test, the indirect hemagglutination assay, the indirect immunofluorescence test, the microcapsule agglutination test, the indirect enzyme- linked immunosorbent assay for IgM antibodies, the dot-ELISA for IgM, and the LEPTO Stick. However, these methods too have several limitations. Some of these methods still require maintenance of live Leptospira cultures for preparing a broadly reactive antigen. Further, although most patients with leptospirosis develop antibodies during the course of infection, these antibodies, not only need a lag period after the infection to become detectable, but once incited, they stay for a long time, even after the pathogenic organisms have been eliminated. It was found that IgM antibodies were detected by dot-ELISA in patients up to the sixth month, decreasing to 57% by the tenth month, and persisting in some patients beyond the twelfth month. This implies that the antibody detection assay is less sensitive during the early period of infection as IgM antibodies were not detected in most patients during the first few days of acute phase of illness and also it cannot be used for monitoring the efficacy of the treatment.

The currently employed serological testing technique also suffers from limitations. A major limitation is the requirement for maintaining living cultures of several serovars and the difficult result interpretation for a current or recent infection as this technique can detect the infection only after a lag period of 7-10 days after which any subject is infected, it takes about 7-10 days for invoking the serologically identifiable reactions. Thus, there is a need for a more accurate, rapid and simpler means for detecting the leptospiral DNA in a given sample.

SUMMARY OF THE INVENTION Thererfore, in one aspect the present invention provides oligonucleotide primer sequences for detection of the presence of Leptospira species in biological or environmental samples.

Accordingly, in one embodiment the present invention provides oligonucleotides ί

sequences to be used as primer for detection of leptospiral DNA in a given sample using a molecular protocol for example a polymerase chain reaction (PCR) technique.

In another aspect the present invention provides a method for detecting leptospiral DNA in a biological or environmental sample with the help of primer sequences of the present invention using molecular protocol for example PCR technique. In one embodiment the present invention provides a method for detecting leptospiral DNA in a given sample, wherein the method comprises of amplifying a segment of leptospiral DNA by subjecting the primers of the present invention specific for leptospires, in combination with heat-stable DNA polymerase in the presence of sample DNA to temperature cycles and detecting the amplified DNA. In yet another aspect the present invention provides a kit for detecting leptospiral DNA in a biological or environmental sample, wherein kit comprises primer sequences of the present invention and optionally other reagents packaged suitably.

In still another aspect the present invention provides a method for detecting leptospiral DNA in a biological sample with the help of primer sequences of the present invention using molecular protocol for example PCR technique for early diagnosis of leptospirosis disease in humans and animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shows a complete nucleotide sequence, SEQ ID NO: 1, a forward primer PI. FIG. 2. Shows a complete nucleotide sequence, SEQ ID NO: 2, a reverse primer P2. FIG. 3. Is a gel photo showing gel electrophoresis of DNA fragments amplified by the PCR using primer set mix comprising forward primer PI (of 24 bases - 5^' AGC ATA CTA TCT CTA TGT TTG GAT having SEQ ID NO. 1) and a reverse primer P2 (of 24 bases - 5^' CCA ACA GAT GCA ACG AAA GAT CCT having SEQ ID NO. 2). Band seen in lane 6 confirms the Leptospirosis infection in the blood sample of the infected person. Band seen in lane 8 confirms the Leptospirosis infection in the urine sample of the infected person.

The gel agarose photo shows:

Lane 1 : negative control : negative

Lane 2 : Healthy negative : negative

Lane 3 : negative control : negative

Lane 4 : Positive nicobar DNA control : positive

Lane 5 : water negative control : negative

Lane 6 : Leptospirosis positive blood sample : positive Lane 7 : water negative control : negative

Lane 8 : Leptospirosis positive Urine : positive

Lane 9 : neat primer 1 - 2 ul.

Lane 10 : neat primer 2- 2 ul.

FIG. 4. Is a gel photo showing gel electrophoresis of DNA fragments amplified by

PCR using primer set mix comprising forward primer PI (of 24 bases - 5^' AGC ATA

CTA TCT CTA TGT TTG GAT having SEQ ID NO. 1) and a reverse primer P2 (of 24 bases - 5^" CCA ACA GAT GCA ACG AAA GAT CCT having SEQ ID NO. 2). Band seen in lane 6 confirms the Leptospirosis infection in the blood sample of the infected person. Band seen in lane 2 confirms the Leptospirosis infection in the serum sample of the infected person.

The gel agarose photo shows:

Lane 1 : Positive nicobar DNA control : positive

Lane 2 : Lepto positive serum : positive

Lane 3 : Malarial positive DNA : negative

Lane 4 : HBV positive DNA : negative

Lane 5 : DNA from serum loaded as such : intact genomic DNA seen.

Lane 6 : water negative control : negative

Lane 7 : healthy negative DNA 1: negative

Lane 8: healthy negative DNA 1: negative

FIG. 5. Is a gel photo showing gel electrophoresis of PCR product 2ul loaded on 1.5% Agarose gel.

The gel agarose photo shows:

Lane Ml: StepUpTMlOO bp ladder (Cat* 612652670501730) Lane 1-7: Sample 1-7 Respectively

Lane M2: StepUpTM500 bp ladder (Cat# 612651970501730)

FIG. 6. Shows T Vector showing Nco I sites.

FIG. 7. Is a gel photo showing gel electrophoresis of showing screening of clones by restriction digestion with Ncol, wherein R= ~3.0kb Vector backbone and S= ~450bp Release.

The gel agarose photo shows:

Lane 1-12: T Clones

Lane M: StepUpTM500 bp ladder (Cat# 612651970501730) FIG. 8. Shows a blast nucleotide sequence of the amplified product as per SEQ ID NO: 3. It shows that amplified product belonged to Leptospira interrogans serovar Pomona with 97% homology.

FIG. 9. : Shows nucleotide sequence of Leptospira interrogans serovar Pomona, SEQ ID NO. 4. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed towards providing oligonucleotide primer sequences for the detection of Leptospira species in biological and environmental samples. The oligonucleotide primer sequences of the present invention are . . capable of detecting DNA of Leptospira species using molecular protocol. In one embodiment the present invention provides oligonucleotides sequences to be used as primers for detection of leptospira species DNA interchangeably referred as leptospiral DNA in a given biological or environmental sample. In another embodiment the present invention provides oligonucleotides sequences to be used as primer for detection of leptospiral DNA in a given sample using a molecular protocol for example a polymerase chain reaction (PCR) technique.

The oligonucleotides sequences to be used as a primer set are designed for the identification of the leptospiral DNA in-vitro. The invented sequences are designed to have the broadest detection profile by remaining specific to the Leptospira genome.

The oligonucleotides sequences have at least 70%, preferably atleast 90% homology with NCBI reference sequences including AM937000.1, DQ286418.1, AY776294.1, AY609332.1, AY609329.1, AY609327.1, AY609325.1, AY609324.1, AY609321.1, AY461909.1, AY461908.1, AY461907.1, AY461905.1, AY461903.1, AY461902.1, AY568679.1, AY442332.1, DQ149595.1, AE010300.1, AF366366.1, U89708.1, AF245281.1, AB094436.2, AB094435.2, AB094433.2, AE016823.1, DQ343231.1, AY776293.1, AY609333.1, AY609328.1, AY609326.1, AY461920.1, AY461910.1, AY461906.1, AY461904.1, AY461901.1, AY423075.1, DQ092412.1, AF181553.1, AJ580493.1, AB094437.2, AB094434.2, AY223718.1. Thus, oligonucleotides sequences to be used as primer sets have a very broad detection profile.

In one preferred embodiment the oligonucleotides sequences comprise of primer set including a forward primer PI : 5^" AGC ATA CTA TCT CTA TGT TTG GAT (24 mer) having SEQ ID NO. 1 and a reverse primer P2: 5" CCA ACA GAT GCA ACG AAA GAT CCT (24 mer) having SEQ ID NO. 2. These oligonucleotides sequences are specific to leptospira LipL32 gene. This primer set has been designed by studying the leptospira genome and the best match of DNA nucleotides that give 40%: 60% with respect to the A+T: G+C.

The oligonucleotides sequences to be used as a primer set as per the present invention can be designed so as to detect only leptospira DNA in a sample from varied sources. The primer set of the present invention can be employed to detect leptospiral DNA obtained by any standard isolation / purification methods. Preferably the primer set is used in any molecular protocol for example a polymerase chain reaction (PCR) technique for detecting leptospira species with specificity. PCR technique is also preferable for detecting leptospira species in biological samples for early diagnosis of DNA in a human and animal. Moreover, the oligonucleotides sequences do not provide false positives hence are more accurate in detecting leptospira species compared to the currently employed methods.

The oligonucleotides sequences can also be used as a primer set to detect only pathogenic leptospira species in a given sample. Further, the oligonucleotides sequences can be extended so as to identify the different species of Leptospira genus in a given sample.

In one embodiment the present invention also provides a method for detecting Leptospiral DNA in a given sample by employing the oligonucleotides sequences of the present invention as a primer set. Preferably the polymerase chain reaction (PCR) is used to detect leptospiral DNA in clinical samples.

In one preferred embodiment the present invention provides a method for detecting Leptospiral DNA in a given sample, wherein the method comprises of amplifying a segment of leptospiral DNA by subjecting primer set comprising a forward primer having SEQ ID NO. 1 and a reverse primer having SEQ ID NO. 2, in combination with heat-stable DNA polymerase in the presence of sample DNA to temperature cycles, and detecting the amplified DNA.

The amplified DNA can be relatively easily detected with the help of non-denaturing gel electrophoresis.

The method optionally comprises subsequent or concomitant hybridization of amplified DNA with labeled probe. Such labeled probes, makes various highly specific methods of detection for example including fluorography, chromatography, autoradiography on solutions or on blots, possible during or after the PCR. Sample DNA can be obtained from biological sample or environmental samples. The biological samples from which the leptospiral DNA can be isolated for example include blood, urine, cerebrospinal fluid. The environmental samples from which the leptospiral DNA can be isolated for example include a water sample suspected to be contaminated with leptospira species. The leptospiral DNA can be isolated from a biological or an environmental sample by following a standard DNA isolation protocol known to a person skilled in the art.

The specificity and high sensitivity of this method in detecting leptospiral DNA provides a valuable tool for detecting the contamination of environmental samples with the leptospira species and in the diagnosis of leptospirosis. In one embodiment the present invention provides a method for diagnosis of leptospirosis in a given subject for example human or animal, wherein the method comprises of amplifying a segment of leptospiral DNA by subjecting primer set comprising a forward primer having SEQ ID NO. 1 and a reverse primer having SEQ ID NO. 2, in combination with heat-stable DNA polymerase in the presence of sample DNA to temperature cycles, and detecting the amplified DNA.

In another embodiment the present invention also provides a kit for detecting leptospiral DNA in a given sample, wherein the kit comprises primer sequences of the present invention and optionally other reagents packaged suitably.

In one embodiment, the kit comprises a ready mix of primer set comprising a forward primer having SEQ ID NO. 1 and a reverse primer having SEQ ID NO. 2 and other reagents provided in suitable containers.

In a preferred embodiment, the kit comprises the ready mix of primer set comprising a forward primer having SEQ ID NO. 1 and a reverse primer having SEQ ID NO. 2, PCR buffer, MgCI2 solution, dNTP mix, PCR buffer and Taq DNA polymerase, positive control and negative control. Such a kit may be used for detecting leptospiral DNA in a given sample by any molecular methods, for example PCR. The ready reaction mixture is to be mixed with the DNA sample for amplification of the primer sequences of the present invention.

The oligonucleotides sequences to be used as a primer, the method and kit as provided by the present invention can detect the presence of leptospiral DNA in a given sample. Such detection can be used to confirm the presence of leptospira species in a given sample for example a water sample suspected to be contaminated with leptospira species. Also, the detection can be used for diagnosis in the early phase of the disease, when bacteria may be present and before antibody titres are at detectable levels with no false positives. The primers can detect genomic species of leptospira which is a group of Leptospiraceae serovars whose DNAs show 70% or more homology at the optimal reassociation temperature of 55^eC or 60% or more homology at a stringent reassociation temperature of 70^0 and in which the related DNAs contain 5% or less unpaired bases. The primer set of the preferred embodiment is very specific towards leptospira species and hence and can detect its presence in wide variety of samples including blood, serum and urine for confirming the infection.

PCR can be used either alone for the detection or in conjunction with other methods such as MAT & ELISA for confirmation.

The present invention is described specifically according to the following examples; however the present invention is not restricted to the said Examples.

EXAMPLES

Example 1:

DNA was isolated from blood of leptospira infected patients pre- confirmed by tri dot test using isolation method as reported in (A quantitative PCR (TaqMan) assay for pathogenic leptospira spp), Lee D Smythe, et al. BMC Infectious Diseases (2002).

The isolated DNA was amplified using the primer sequences having SEQ ID. 1 and SEQ ID. 2 of the present invention by the PCR technique as follows:

Amplification of DNA from blood of infected patient was carried out with primer mix having final conc.0.5uM, dNTP mix 250uM, taq polymerase 2.5u. PCR was carried out in a thermal cycler program, where the initial denaturation step was carried out at 95 degree Centigrade for 5 mins. The DNA denaturation step was carried out at 94 degree Centigrade for 30 sec followed by primer -DNA template annealing at 55oC for 30 sec and nucleotide polymerization for 72 degree Centigrade for 30 sec for 35 cycles. Final extension at 72 degree Centigrade for 5 mins and Hold time 4 degree Centigrade till infinity. Bands of Leptospiral DNA were analyzed on agarose gel by comparing with positive control and DNA ladder 100 base pair.

Band in lane 6 as seen in FIG. 3 confirms the leptospirosis infection. Parallel run with healthy negative blood was also analyzed on gel electrophoresis, herein confirming no false negatives as seen in FIG. 3.

Example 2: DNA was isolated from urine of leptospira infected patients pre- confirmed by tri dot test using the isolation method as reported in (A quantitative PCR (TaqMan) assay for pathogenic leptospira spp), Lee D Smythe, et al. BMC Infectious Diseases (2002).

The isolated DNA was amplified using the primer sequences having SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention by the PCR technique as per Example 1.

Band in lane 8 as seen in FIG. 3 confirms the leptospirosis infection. Parallel run with healthy negative blood was also analyzed on gel electrophoresis, herein confirming no false negatives as seen in FIG. 3.

Example 3: DNA was isolated from serum of leptospira infected patients pre- confirmed by tri dot test using the isolation method as reported in (A quantitative PCR (TaqMan) assay for pathogenic leptospira spp), Lee D Smythe, et al. BMC Infectious Diseases (2002).

The isolated DNA was amplified using the primer sequences having SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention by the PCR technique as per Example 1. Band ii lane 2 as seen in FIG. 4 confirms the leptospirosis infection. Parallel run with positive malaria and HBV blood was also analyzed on gel electrophoresis, herein confirming no false positives as seen in FIG. 4.

Example 4:

Specificity of primer binding to leptospirosis genome: In order to confirm the specificity of primer binding to leptospirosis genome cloning was performed. The Amplified PCR products (post PCR) were checked on 2.0% agarose gel as seen in FIG. 5. PCR product were cloned in T Vector within Ncol sites as depicted in FIG. 6. Ncol sites (CCATGG) on the vector are highlighted on both forward and reverse sequence data (FIG. 6). The clones were confirmed by release of insert using Ncol restriction endonuclease. The cloned DNA samples were sequenced bi-directionally using T7 forward & SP6 reverse primers (vector specific primers).

Cloning of PCR product into T Vector :

Ligation was performed by using 3.0ul of PCR product and proceeded for transformation into DH5ctcells.

Screening of clones by restriction digestion with Ncol: The plasmids were digested with Ncol Restriction endonuclease for the confirmation of clones. Digestion confirmed clones were bi-directionally sequenced with T7 and SP6 primers (FIG. 7).

The blast sequence showed that the amplified product belonged to Leptospira interrogans serovar Pomona with 97% homology as seen in FIG. 8 and SEQ. ID No. 3. The gene sequence of Leptospira interrogans serovar Pomona is presented in FIG. 9 and SEQ ID NO. 4.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application

Claims

1. A set of primer sequences for detection of leptospira species DNA in a given biological or environmental sample using PCR technique, a forward primer having a SEQ ID NO. 1 and a reverse primer P2 having a SEQ ID NO. 2.

2. The set of primer as claimed in claim 1, wherein primer sequences are specific to leptospira LipL32 gene.

3. A method for detecting leptospiral DNA in a given sample, wherein the method comprises of amplifying a segment of leptospiral DNA by subjecting the primers of claim 1, in combination with heat-stable DNA polymerase in the presence of sample DNA to temperature cycles and detecting the amplified DNA.

4. The method as claimed in claim 3, wherein the amplified DNA is detected by non-denaturing gel electrophoresis.

5. The method as claimed in claim 3, optionally comprises subsequent or concomitant hybridization of amplified DNA with labeled probe during or after the PCR.

6. The method as claimed in claim 5, wherein labeled DNA is detected by method including fluorography, chromatography, autoradiography on solutions or on blots.

7. A kit for detecting leptospira species DNA, comprising a set of primers as claimed in claim 1 and optionally other reagents packaged suitably.

8. The kit as claimed in claim 7, wherein said other reagents include PCR buffer, MgCI2 solution, dNTP mix, PCR buffer and Taq DNA polymerase, positive control and negative control.

9. A method for diagnosis of leptospirosis in a given subject including human and animal, wherein the method comprises of amplifying a segment of leptospiral DNA by subjecting a set of primers of claim 1, in combination with heat-stable DNA polymerase in the presence of sample DNA to temperature cycles, and detecting the amplified DNA.

10. The method as claimed in claim 9, wherein the amplified DNA is detected by non-denaturing gel electrophoresis.

11. The method as claimed in claim 9, optionally comprises subsequent or concomitant hybridization of amplified DNA with labeled probe during or after the PCR.

12. The method as claimed in claim 11, wherein labeled DNA is detected by method including fluorography, chromatography, autoradiography on solutions or on blots.