Claims:We Claim:
1. A Method for the Detection and Control of Leptospira comprising steps of:
(a) Processing the biological sample to obtain DNA of the parasite by isolating them from the blood, serum and other human bodily fluids;
(b) Subjecting the DNA to polymerase chain reaction using primers specific for Leptospira in a thermal cycler or isothermally , followed by analyzing the amplicons on agarose gel and/or real time, or nanotechnology based on gold or silver particles or biosensors or polynucleotide sequencing to identify the region exhibiting significant similarity;
(c) The real-time polymerase chain reaction method is carried out by a holding period of 2 minutes at 50°C, initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and at 60°C for 1 minute for the target LMLS90.
(d) In polymerase chain reaction the targets LMLS90 were subjected to initial denaturation at 95°C for 5 minutes and then 35 cycles of 95°C for 1 minute, 48°C for 1 minute, and 72°C for 1 minute 30 seconds, followed by final extension at 72°C for 10 minutes. The products were run on 2% agarose (Sigma) gels in 1× TBE buffer (8.9 mM Tris, 8.9 mM Boric acid, 2 mM EDTA).
(e) In real-time polymerase chain reaction is carried out by holding period of 2 minutes at 50°C, initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and at 60°C for 1 minute for the target LMLS90, then the 10µl polymerase chain reaction mixtures were subjected to real-time amplification in a 7500 Fast Real-Time PCR System (Applied Biosystems), the reaction mixture consisted of 3.75µl of plasmid DNA of LMLS90, 18.75µl of 2x SYBR green master-mix, 1.5µl of each forward and reverse primers (15 pmol each) and 12µl of milliQ water.
(f) This mixture then divided into triplicates by taking 10µl each in real-time plate and performed with a holding period of 2 minutes at 50°C, initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and at 60°C for 1 minute and each specimen is made to run in triplicates for the real-time PCR assay.
(g) The end product of polymerase chain reaction with significant specificity to Leptospira is identified on the basis of gel electrophoresis and sequencing data analysis and standard values.
(h) Control of the disease through screening of subjects in endemic area through this rapid method to provide preventive measures; and in non-endemic areas for suspected leptospirosis.
2. A method as claimed in claim 1, wherein the method of polymerase chain reaction can be carried out using real-time thermal cycler and associated components.
3. A kit comprising:
(a) Component A comprising a set of polynucleotides specific to Leptospira;
(b) Component B comprising reagents for Polymerase chain reaction;
(c) Component C comprising a positive control; and instructions, wherein the instructions outline in detail the method as claimed in Claim 1.
, Description:FIELD OF INVENTION:
A method for detection and control of Leptospira infection in humans
BACKGROUND OF INVENTION:
Leptospirosis is emerging and re-emerging disease affecting humans, animals and environment causing socio-economic loss as well as threat to lives. Every year more than 500,000 severe human cases of leptospirosis are estimated globally. Yet, leptospirosis is considered as neglected tropical disease and in India, lacks awareness because of unreported data. However, of late, increasing numbers of outbreak of leptospirosis have been reported from Kerala, Gujarat, Tamil Nadu, Karnataka and other parts of India. The warm and humid climatic condition of Southern India favors the growth and survival of its causative agent, Leptospira species. Hence, there is a need for effective diagnosis and control of leptospirosis. In symptomatic and asymptomatic infections, a sensitive, accurate and rapid diagnostic tool is essential to treat leptospirosis. There is lacunae in the diagnostic field to identify parasites in acute, chronic and latent (present and post infection) form of leptospirosis in humans; and in its mild and moderate forms.
Prior art search revealed that most of the patented (WO20010647763A1, US20150004623, WO2008108510A1) diagnostic methods developed to detect Leptospira rely on the detection of one or other specific protein, against which different types of antibodies are raised for detection. But such methods rely on the presence of the specific protein in a patient’s sample and have the drawback of being useful only after a few days of infection (minimum two weeks), by which time the parasite may have caused some damage to the host. In addition, in such methods incorrect storage of the kits/reagents, or poor sample preparation could lead to contamination, leading to false positive or false negative results. Tests based on antibody detection are prone to non-specific binding upon over-incubation, leading to false positive results. In case the gene expressing the protein against which the antibody has been raise is deleted, the results can be misleading and lead to misdiagnosis. The reported specificity and sensitivity of such methods are in the range between 70-90%. The present method utilizes detection of specific nucleotide sequences in the genome of the organism that are present in high copy numbers, which enhances the detection of the pathogen, even if present in low numbers.
OBJECT OF THE INVENTION:
The principal object of this invention is to provide a highly specific diagnostic method for the detection and control of leptospirosis in humans
A further object of this invention is to preempt the infection prior to the disease through sensitive diagnosis.
Another object of this invention is to provide methods to identify and detect the duration of the leptospirosis disease for treatment and management purposes.
It is another object of the present disclosure to provide a set of polynucleotide sequences (LMLS90) that are specific for Leptospira interrogans exhibiting significant sensitivity, and suitable for detecting early leptospirosis.
It is yet another object of the present disclosure to provide a method of detecting Leptospira interrogans which is specific, sensitive, and accurate.
It is still another object of the present disclosure to provide a method to identify 90 bases unique to Leptospira interrogans genome, present on chromosome 1.
It is yet another object of the present disclosure to provide an efficient method which reduces the complexity of the DNA fragment mixture by shearing, and amplifies a specific region of the DNA obtained using blood, serum, saliva, stool and urine of humans suspected with leptospirosis.
SUMMARY OF THE INVENTION:
The present invention provides a species-specific, sensitive polymerase chain reaction assay for the detection of Leptospira in blood, which can aid in leptospirosis diagnosis and treatment.
The present invention discloses a method to identify the species-specific parasite, an intra- and inter-genic, multi-locus stretch of 90 nucleotides exclusive in Leptospira interrogans that can be utilized in isothermal or higher temperature DNA amplification reaction for detecting leptospirosis.
Present invention also claims its application in other methods of detection such as isothermal reaction, cold reaction, nanotechnology-based detection, biosensor-based detection, peptide nucleic acid detection, or any another reactions which primarily use disclosed polynucleotides to facilitate the disease detection.
DETAILED DESCRIPITON OF INVENTION:
The present disclosure is suitable to identify individuals infected with leptospirosis at an early stage or during various stages of disease in an endemic region or otherwise. The present disclosure is also suitable for the rapid detection of very low levels of parasite in the blood.
An extensive and comparative polynucleotide analysis of Leptospira genome by using bioinformatics tools such as clustalW, BioEdit and Blast 2.2.27 release (blastN (nr/nt)) http://blast.ncbi.nlm.nih.gov/Blast.cgi# with an E-value cutoff range of (1E-10) revealed 70 copies (Chr1: 1897-1985) of 90 nucleotides sequences with 75 – 100% homology in the genome.
Primers were designed manually for candidate target sequence (LMLS90) by using Primer3 Input version 0.4.0 (http://frodo.wi.mit.edu/primer3/) to amplify the 90bp region of Leptospira genome (forward – 5’-AACAAGGCACATTCTTCAA-3’ and reverse – 5’-GTTTTCTTTATCAGAACAACATACT-3’).
Primers of varying length, composition, modifications were identified that can amplify the specific region at a range of conditions, with source materials from human subjects such as whole blood, serum, saliva, urine and stool and other bodily sources of the parasite, and method of processing these samples, through which the parasite can be detected. In order to avoid other genome sequences, cross reaction target sequence (LMLS90) were extensively compared with all the genome sequences (NCBI) with BLAST 2.2.26 release and no significant similarity was found. Comparisons were also made with human genome by using UCSC BLAT (RefSeq, Primary Reference Assembly, Feb. 2009 (GRCh37/hg19)) and no significant similarity was found.
A unique sequence is displayed, present up to 70 times in the complete genomes of Leptospira with overall homology alignment of 75 – 100%, and is more sensitive than any other targets when utilized in a single-step amplification-based method for the detection of the aforementioned species. Primers were able to amplify varying lengths of LMLS90 sequences and were successful in detecting parasite levels of up to 0.1 copy/10µl of blood sample.