WO2011001449A2

WO2011001449A2 - Primers, pcr reaction mixture and methods thereof

Info

Publication number: WO2011001449A2
Application number: PCT/IN2010/000421
Authority: WO
Inventors: Dipshikha Chakravortty; Arvindhan Govindaswamy Nagarajan
Original assignee: Indian Institute Of Science
Priority date: 2009-06-18
Filing date: 2010-06-18
Publication date: 2011-01-06
Also published as: WO2011001449A3

Abstract

The present disclosure relates to primers for amplification of genes of Salmonella species. The present disclosure gives a detailed description of methods for determining the presence of Salmonella species in various sources like blood/serum/plasma samples by employing specific nucleotide primers. The designed primers are used in conjugation with a PCR reaction mixture. The disclosure also elaborates on a method to obtain a template for PCR.

Description

"PRIMERS, PCR REACTION MIXTURE AND METHODS THEREOF"

TECHNICAL FIELD

The present disclosure is in relation to a method for the detection of Salmonella infection by employing primers and primers per se. The present disclosure further relates to a PCR reaction mixture and a method used to obtain a template for PCR.

BACKGROUND OFTHE DISCLOSURE

Salmonella enterica is an important enteric pathogen and is involved in causing both systemic and intestinal diseases in humans and a wide range of other hosts. Serotypes within subspecies I {Salmonella enterica subsp. enterica) are responsible for the vast majority of salmonellosis infections in warm-blooded animals. S. typhi and 5". paratyphi

A causes typhoid fever strictly in humans mostly in developing countries, with no age exemption but it is less common before 2 years. According to an estimate the worldwide incidence of typhoid fever is 16 million cases annually and death rate is 6 lakhs individuals per year. According to a Press Information Bureau, Government of

India, release dated February 22, 2006, the morbidity due to typhoid varies from 102 to

2219/100,000 population in different parts of India and in some areas typhoid fever is responsible for 2-5% of all deaths. The problem of typhoid fever has been exacerbated by the appearance of multiple drug resistant strains, the treatment of which would depend on newer antibiotics and early and precise diagnosis.

The existing modes of diagnosis are through detection of antibodies against Salmonella through WIDAL test and other serological tests like DOT enzyme immunoassay, dip stick assays and semi-quantitative tube agglutination test. Apart from this, the bacteraemia observed in typhoid around day 6-9 enables it to be detected through blood culture test and PCR amplification of bacterial DNA from blood. Among the commonly available diagnostic tests, WEDAL test and other serological diagnostic methods have their limitation because of the low specificity of the test. There are reports of large number of false positives especially in typhoid endemic areas and among patients who have had typhoid fever previously. Blood culture test has the major disadvantage of being a time consuming test which takes 2 to 3 days. PCR based diagnoses are superior to classical serological method, WIDAL test and blood culture test in terms of their specificity and sensitivity. The PCR based systems currently use primers against flagellin genes, MA, invA and spvC genes. The varying distribution of invA, and spvC genes among Salmonella isolates from animals highlights the unsuitability of these two genes as PCR probes for Salmonella detection. The present disclosure overcomes the limitation associated with the prior art.

STATEMENT OF DISCLOSURE

Accordingly the present disclosure relates to Primers of SEQ ID Nos. 1, 2, 3 and 4; a PCR reaction mixture, said mixture comprising of Tris having a concentration preferably of about 67 raM and pH preferably of about 8.8, Ammonium Sulphate having a concentration preferably of about 16 raM, polyethylene glycol p-(l,l,3,3- tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.1%, Magnesium Chloride having a concentration preferably of about 2.5 mM, dNTPs having a concentration preferably of about 200 μM, primers as set forth in SEQ ID Nos. 1, 2, 3 and 4, each having a concentration preferably of about 25 picomoles and Taq polymerase preferably about 2units; a method of obtaining a template for PCR, said method comprising steps of- a) centrifuging blood sample to obtain a pellet, b) adding polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether to the pellet and centrifuging to obtain another pellet, c) washing the centrifuged pellet with the polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether and d) suspending the washed pellet in nuclease free water followed by boiling the entire volume, to obtain the template for PCR; a method of detecting Salmonella infection, said method comprising steps of- a) forming a PCR reaction mixture, b) adding sample to be detected and primers selected from a group comprising SEQ ID Nos. 1 and 2, and 3 and 4, individually or in combination and c) subjecting the reaction mixture to PCR to obtain copies of target nucleotide sequence followed by gel electrophoresis to detect the Salmonella infection; and a kit for detection of Salmonella, wherein said kit comprises primers of SEQ ID Nos. 1, 2, 3 and 4 along with the PCR reaction mixture.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

'

Figure 1 shows a schematic diagram of the genome organization of the loci used for primer designing in the three Salmonella strains S. typhi CTl 8 (STY CTl 8), S. paratyphi A (SPA), S. typhi Ty2 (STY Ty2). Row 1 shows the expected size of the PCR product. Hollow arrows in row 2 depict the primer binding sites of the two sets of primers. The genome organizations of the three serovars are depicted as solid arrows, each representing a gene with its GenBank locus tag number given below. Dotted lines represent genes which are absent in a particular serovar.

Figure 2 shows that the PCR detection is specific for Salmonella and differentiates various typhoid causing serovars. Colony PCR was performed for different serovars of

Salmonella and other pathogenic and non-pathogenic bacteria. PCR amplification was performed with the two sets of primers for 30 cycles and the product was run on 1.5 % agarose gel. The samples have been loaded on the gel in the same order as in Table 1.

The 384 bp band is the amplification product of the gene STY0312 of S. typhi CT18 and its homolog in S. paratyphi A and is found in both the serovars. The 1043 bp band is the amplification product of the loci spanning the genes STY0313 to STY0316 and their homologs in S. typhi Ty2. This loci is absent in S. paratyphi A. Both these bands are absent in all the other bacterial strains tested. Figure 3 shows that the genomic loci used as diagnostic marker is potentially stable. 20 clinical isolates from Pondicherry (South India) and 12 clinical isolates from Nagpur (Central India) were analyzed by colony PCR to show that the region is potentially stable. The amplified products were run on 0.8 % agarose gel. (A) The 20 clinical isolates showed two bands, characteristic of S. typhi CTl 8 which has also been confirmed through serotyping. (B) This representative gel shows 7 samples out of 12 samples obtained from Nagpur. Among 7 samples, three samples showed only the lower 384 bp band of S. paratyphi A (2,3,5) and 4 samples showed two bands of S. typhi CT18 (1,4,6,7). Samples 8, 9 and 10 are controls with isolated colonies of S. paratyphi A, S. typhi CTl 8 and S. typhi Ty2 respectively.

Figure 4 shows the PCR based assay can detect as low as one bacterium. The minimum number of bacteria that can be detected by this method was determined by diluting 0.3 O.D (600nm) adjusted culture which contains 1.5 x 10⁸ bacteria to various dilutions in PBS and sterile blood. They were then subjected to one round of PCR amplification for 35 cycles; the products were then visualized by running on 0.8 % agarose gel. The corresponding number of bacteria/ml is given at the top of each lane. (A) Varying number of S. typhi CTl 8 diluted in PBS showing that one bacteria/ml can produce a visible band. (B) Varying number of S. typhi CTl 8 diluted in blood, shows that the procedure can detect as low as four bacteria/ml of blood. (C) Varying number of S. paratyphi A diluted in PBS, shows a sensitivity of detection of four bacteria/ml. (D) Varying number of S. paratyphi A diluted in blood, shows a lower detection limit of one bacterium/ml of blood.

Figure 5 shows the PCR assay is more sensitive than WIDAL test. Two representative gel pictures showing the PCR products amplified from patient blood samples. The corresponding WIDAL result (W) are given as +/- above each sample respectively. (A) Lanes 1, 2 and 3 are samples positive for S. paratyphi A. Lanes 4-13 are samples positive for S. typhi. (B) Lanes 1, 4, 6, 7, 8, 9 are samples positive for S .typhi CTl 8. Lanes 2, 3, 10 and 11 are PCR negative samples. Lane 5 is PCR positive for S. typhi Ty2. Lane 'C is positive control for S. typhi CT18. The PCR assay is 40 % more sensitive than WIDAL test in this given partial sample data.

DETAILED DESCRIPTION OF DISCLOSURE

The present disclosure relates to Primers of SEQ ID Nos. 1, 2, 3 and 4.

In an embodiment of the present disclosure the primers having SEQ ID Nos. 1 and 3 are forward primers and the primers having SEQ ID Nos. 2 and 4 are reverse primers and, the primers form a combination set forth as SEQ ID Nos. 1 and 2, and SEQ ID Nos. 3 and 4.

In another embodiment of the present disclosure, the primers having SEQ ID Nos. 1 and 2 correspond to genomic loci STY0312 in Salmonella typhi CTl 8 and SPA2476 in Salmonella paratyphi A respectively and, wherein SEQ ED Nos. 3 corresponds to STY0313 in Salmonella typhi CTl 8, SPA2475 in Salmonella paratyphi A and t2576 in Salmonella typhi Ty2 respectively and, wherein SEQ ID No. 4 corresponds to STY0316 in Salmonella typhi CT 18 and Salmonella typhi Ty2 respectively.

The present disclosure relates to a PCR reaction mixture, said mixture comprising of Tris having a concentration preferably of about 67 mM and pH preferably of about 8.8, Ammonium Sulphate having a concentration preferably of about 16 mM, polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.1%, Magnesium Chloride having a concentration preferably of about 2.5 mM, dNTPs having a concentration preferably of about 200 μM, primers as set forth in SEQ ID Nos. 1, 2, 3 and 4, each having a concentration preferably of about 25 picomoles and Taq polymerase preferably about 2units.

The present disclosure relates to a method of obtaining a template for PCR, said method comprising steps of:

(a) centrifuging blood sample to obtain a pellet;

(b) adding polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether to the pellet and centrifuging to obtain another pellet;

(c) washing the centrifuged pellet with the polyethylene glycol p-( 1,1,3,3- tetramethylbutyl)-phenyl ether; and

(d) suspending the washed pellet in nuclease free water followed by boiling the entire volume, to obtain the template for PCR.

In an embodiment of the present disclosure, the blood sample has a volume ranging from about 200 μl to about 300 μl, preferably about 200 μl, the centrifuging is carried out from about 10,000 g to about 12,000 g, preferably at about 12,000 g for a time duration of about 2 minutes to about 4 minutes, preferably about 2 minutes, the adding of polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.2% and a volume preferably of about 1.0 ml, followed by vortexing, incubating of the pellet at room temperature for a time duration ranging from about 5 minutes to about 10 minutes, preferably about 5 minutes, and re-washing of the pellet with the polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether, the suspending in nuclease-free water of volume ranging from about 15 μl to 30 μl, preferably of about 20 μl and the boiling temperature preferably at about 99 ⁰C for a time duration preferably of about 10 minutes. The present disclosure relates to a method of detecting Salmonella infection, said method comprising steps of:

(a) forming a PCR reaction mixture;

(b) adding sample to be detected and primers selected from a group comprising SEQ ID Nos. 1 and 2, and 3 and 4, individually or in combination; and

(c) subjecting the reaction mixture to PCR to obtain copies of target nucleotide sequence followed by gel electrophoresis to detect the Salmonella infection.

In an embodiment of the present disclosure the PCR reaction mixture comprises Tris having a concentration preferably of about 67 mM and a pH preferably of about 8.8, Ammonium Sulphate having a concentration preferably of about 16 mM, polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.1%, Magnesium Chloride having a concentration preferably of about 2.5 mM, dNTPs having a concentration preferably of about 200 μM, the primers each having a concentration preferably of about 25 picomoles and Taq polymerase preferably about 2units.

In another embodiment of the present disclosure the primers having SEQ ID Nos. 1 and 3 are forward primers and the primers having SEQ ID Nos. 3 and 4 are reverse primers and, the sample is selected from a group comprising blood, serum and plasma.

The present disclosure relates to a kit for detection of Salmonella, wherein said kit comprises primers of SEQ ID Nos. 1, 2, 3 and 4 along with the PCR reaction mixture. In an embodiment of the present disclosure the PCR reaction mixture comprises Tris having a concentration preferably of about 67 mM and a pH preferably of about 8.8, Ammonium Sulphate having a concentration preferably of about 16 mM, polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.1%, Magnesium Chloride having a concentration preferably of about 2.5 mM, dNTPs having a concentration preferably of about 200 μM, the primers each having a concentration preferably of about 25 picomoles and Taq polymerase preferably about 2units. Typhoid fever is becoming an ever increasing threat in the developing countries. The instant disclosure involves considerable improvement upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhi and S. paratyphi A, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphi A. An additional set of primers amplify another region in S. typhi CT 18 and S. typhi Ty2 and are corresponding to the region between the genes STY0313 to STY0316 but which is absent in S. paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region was tested for genomic stability by amplifying them from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers also differentiate between S. typhi CT 18, S. typhi Ty2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% as compared to the WIDAL test which had only 63%. As is observed in the current disclosure, the PCR assay is more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

Many genes encoded by Salmonella pathogenicity islands have evolved differentially in typhoidal and non-typhoidal Salmonella giving rise to different allelic variants of these genes. These genes are present in the different serovars of Salmonella, and their orthologs in other species of bacteria share varying degrees of identity at the nucleotide levels. These differences if minor, at certain PCR conditions can lead to promiscuous amplification, thereby leading to false positive results. This problem can be overcome by choosing those regions which are unique to S. typhi and S. paratyphi A. Though certain pathogenicity islands are unique to S. typhi and S. paratyphi A, like SPI-7 and SPI-8, these islands are known to be unstable. These islands can be excised by the activation of certain recombinases as exemplified by the isolation of clinical variants lacking Salmonella pathogenicity island-7 (SPI-7). Also the presence of gene encoding integrase in the SPI-8 island suggests that it is a mobile island. For the same reason insertion sequences and bacteriophage genes are not good candidates for the use of diagnostic purposes. But a thorough examination of the whole genome sequence of S. typhi, S. paratyphi A and S. typhimurium highlights the existence of genomic regions of unknown function with no homologous genes in related serovars and without the features of mobile DNA sequences. Using these criteria for the identification of a good diagnostic marker gene in S. typhi CTl 8, the present disclosure identifies the genomic loci spanning the genes STY0312, unique to S. typhi CT 18 and S. paratyphi, the causative agents of typhoid. The adjoining loci spanning STY0313 to STY0316 was different in S. typhi Ty2 and S. paratyphi but otherwise conserved in most Salmonella species. This region is part of SPI-6 which is present in many Salmonella enterica subspecies I. In the present disclosure primers have been designed against these regions to amplify and thereby differentiate the serovars of typhoidal Salmonella.

The present disclosure is further elaborated with the help of following examples and associated figures. However, these examples should not be construed to limit the scope of the present disclosure. Further, as the scope of the present disclosure is non-limiting in nature, the instant primers can be applied to various other biotechnological applications such as but not limiting to those illustrated in the following examples. Further, all the possible amplification experiments involved in all such biotechnological applications fall under the purview of the present disclosure.

EXAMPLE 1

BACTERIAL STRAINS AND CULTURE CONDITIONS

The different bacterial strains used in this study are given in Table 1. Routine culturing of bacteria is done in LB broth and in LB Agar plate at a temperature of about 37 ⁰C. During blood culture diagnosis the bacteria is plated on Salmonella-Shigella agar (Hi- Media), selective media for Salmonella species. S. typhimurium LT2 strain is nalidixic acid resistant and 50 μg/ml of nalidixic acid was added to LB during its culturing.

Table 1

BLOOD COLLECTION

One ml of blood sample is collected from healthy donors and typhoid suspected patients with EDTA. The samples are collected before antibiotic treatment. In the present disclosure, the samples have been obtained from Indian Institute of Science, Health centre and R.V diagnostic centre, Bangalore, according to the Institute's human ethics committee. A total of 50 patient's blood samples were collected.

EXAMPLE 2

PROCESSING OF BLOOD SAMPLES FOR PCR

Artificially inoculated blood samples are made by adding various amounts of bacteria from serially diluted cultures of 0.3 O.D (600nm). Then the samples are processed as mentioned below and PCR is done using the specific primers as set forth in Sequence ID Nos. 1, 2, 3 and 4.

PREPARATION OF TEMPLATES FOR PCR

Templates for PCR are prepared from both patient blood samples, and healthy donor blood samples artificially inoculated with S. typhi and S. paratyphi A. For clinical samples, one ml of blood is centrifuged at 10,000 x g for 5 min, to obtain a pellet. 1 ml of 0.2 % Triton X-100 or polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether, is added to the pellet, vortexed and incubated for 10 min at room temperature followed by centrifugation at 10,000 x g for 10 min. The supernatant is decanted and thereafter washing of the pellet with 0.2 % Triton X-100 is repeated. The final pellet is again washed with 1 ml of Nuclease free water. Finally, the washed pellet is resuspended in 30 μl of Nuclease free water. This pellet is boiled for a specific duration of time and the entire volume is used as template for the PCR reaction.

Triton-X lyses mammalian cells by disrupting the lipid bilayer. This helps in release of intracellular bacteria from the infected macrophages. At the same time 0.1% Triton-X- 100 does not lyse the bacterial cells. Hence, the samples are boiled. Boiling the sample helps in lysis of the bacteria and release of DNA into the lysate which acts as the template for PCR detection.

EXAMPLE 3

BIOCHEMICAL TESTS

1. BLOOD CULTURE TEST

2.5 ml of patient's blood is drawn and added to brain heart infusion media. It is then incubated at 37 ⁰C for 24 h and later streaked onto MacConkey agar plate. If the colony is non-lactose fermenting then further oxidase test and slide agglutination test is done. Those which are negative for oxidase test and positive for slide-agglutination test are further confirmed through biochemical tests where S. typhi is indole negative, urease negative and mannitol non- fermenter.

2. SLIDE AGGLUTINATION TEST On a slide, 2-3 drops of saline is added and the test colony is picked from the growth plate and thoroughly mixed with the saline. To this mixture one drop of WIDAL positive indicator serum is added. Formation of clumps is an indication of the presence of Salmonella.

. ^"

3. WIDAL TEST

Rapid slide test for qualitative in vitro determination of antibodies in serum against Salmonella typhi O' and 'H' and/or Salmonella paratyphi A(H) and B(H) antigens were done using TyDAL kit (Arsitha Diatech) following the manufacturer's instructions.

EXAMPLE 4

PRIMER DESIGNING

The primers are designed after analyzing the complete genome of S. typhimurium, S. typhi CTl 8, S. typhi Ty2 and S. paratyphi A, available at GenBank NCBI. The above four sequences are analysed using wgVISTA; which is a set of programs for comparing whole genome sequences of two microbes which are less than 10MB along with annotation. The program is implemented as an on-line server which provides access to the whole-genome alignment pipeline. Unique regions present in the systemic typhoid causing organisms, S. typhi and S. paratyphi A but absent in enteric disease causing organism S. typhimurium are identified. The regions of SPI-7 and SPI-8, though unique to S. typhi and S. paratyphi are omitted because of their unstable nature. The gene STY0312 from SPI-6 of S. typhi is found to be present in S. typhi and S. paratyphi A and absent from S. typhimurium though SPI-6 is present in S. typhimurium as Salmonella genomic island (SGI). The genes STY0312, STY0313, STY0314 and STY0316 are subjected to homology searches through BLAST by comparing against all completely and partially sequenced microbial genomes. The gene STY0312 is found in the typhoidal Salmonella serovars S. typhi CTl 8 and S. paratyphi A. Salmonella enterica subsp. enterica serovar Heidelberg str. SL476, Salmonella enterica subsp. enterica serovar Newport str. SL254 and Salmonella enterica subsp. enterica serovar Typhi Ty2 have a partial gene sequence but are not amplified using the given set of primers. The genes STY0313, STY0314 and STY0316 are found in many serovars of Salmonella enterica. subspecies I. From this locus spanning the genes STY0313 to STY0316, unique regions specific to S. typhi CT 18 and S. typhi Ty2 were identified. This region is also present in the non-typhoidal strains Salmonella enterica subsp. serovar Heidelberg str. SL486 and Salmonella enterica subsp. enterica serovar Newport str. SL254.

In other words, the whole genome of Salmonella typhi is compared against other Salmonella species to identify unique loci. The software used for the analysis is wgVISTA. Then the omitting of those regions that are present in the highly mobile pathogenic islands is done, followed by concentrating on the stable genomic loci. This is to make sure that a strain variant lacking the mobile pathogenic islands, which are frequently reported, would still not be able to escape detection. With these considerations, the present study concentrated on the loci in question and designed primers, which can amplify it. The entire loci is analysed by megablast to see for the prevalence of this loci in the various Salmonella serovars and for other rearrangements in that loci if any.

Using S. typhi CT 18 as the reference strain two sets of primers are designed such that they would amplify two genomic loci spanning the gene STY0312 (SPA2476 in S. paratyphi A, absent in S. typhi Ty2) which encodes a putative secreted protein and genes STY0313 (SPA2475 in S. paratyphi A, t2576 in S. typhi Ty2) to STY0316 (not present in S. paratyphi A, t2574 in S. typhi Ty2) in a non-overlapping way. The gene STY0312 is absent in S. typhi Ty2 and the loci spanning STY0313 to STY0316 is absent in S. paratyphi A whereas the entire loci is absent in S. typhimurium. To bring in further clarity, it can be stated that the genomic loci spanning STY0313 to STY0316 is partially truncated in S. paratyphi. The homologous gene of STY0313 is SPA2475 and that of STY0314 is SPA2474. The homologous gene of STY0316 is absent in S.paratyphi. Since the reverse primer binds to this gene, there will be no amplification of this locus, which spans 3 genes. PCR amplification using the two sets of primers with genomic loci of different Salmonella strains as the template would yield two bands of size 1043 and 384 bp for S. typhi CT18, one band of 384 bp size for S. typhi Ty2, one band of 1043 for S. paratyphi A and no band for S. typhimurium as depicted in Figure 1. Salmonella enterica subsp. enterica serovar Typhi CTl 8, complete genome: ACCESSION NC 003198; Salmonella enterica subsp. enterica serovar Typhi Ty2, complete genome: ACCESSION NC 004631; Salmonella enterica subsp. enterica serovar Paratyphi A ATCC 9150, complete genome: ACCESSION NC_006511; Salmonella typhimurium LT2, complete genome: ACCESSION NC 003197

Primer set 1:

STY0312/SPA2476 (F): 5'-ATGTTCAGTA AAATAGTGTC ATTGCTTTTG -3'- SEQ ID No.l

STY0312/SPA2476 (R): 5'- TTGTAGCGCC GGAAATGATA TTCT- 3'- SEQ ID

No.2

Primer set 2:

STY0313/SPA2475/t2576 (F): 5' -CTTGACGTAC CGGTAGAGAT ATACTGGCT- 3'- SEQ ID No.3

STY0316/t2574 (R): 5'-CTTTACATCT GTTCCGCCCC AGGCAAATAC- 3' - SEQ ID No.4 The combination of primers that can be used for diagnosis are the forward and reverse primers of gene STY0312 and the forward primer of STY0313 and the reverse primer binding to the gene STY0316. Though it will be expected that since the genes STY0312-0316 lie in a contiguous sequence the forward primer of gene StyO312 and the reverse primer of gene STY0316 can amplify a product. However, in order to prevent ambiguity arising form multiple bands, this has been avoided. This has been achieved in the instant invention by designing the primers in such a way that the forward primer of STY0312 and the reverse primer of STY0316 bind to the same DNA strand and thus do not give rise to an amplified product. Another embodiment of present disclosure is a kit for detection of Salmonella species, said kit comprising primers for amplification of genes of Salmonella species and the reagents and buffers for amplification. EXAMPLE 5

PCR CONDITIONS

PCR protocol is used for the detection of infection on the clinical samples and artificially inoculated blood samples.. A volume of 200-300 μl of blood is taken and centrifuged at 12,000 x g for 2 min. To the pellet 1 ml of 0.2 % Triton X-100 is added and kept for 5 min. The pellet containing the cell debris and bacteria is centrifuged and the supernatant discarded. The triton wash is repeated and the pellet suspended in 20 μl of Nuclease free water. This pellet is boiled for 10 min at 99 ⁰C and the entire volume is used as template for PCR reaction.

The PCR reaction mixture (50 μl) contained about 67 mM Tris, at pH of about 8.8, about 16 mM (NH₄^SO₄, about 2.5 mM MgCl₂, about 0.1 % Triton X-100, about 200 μM dNTPs, about 25 picomoles of each primer, and about two units of Taq DNA polymerase .

Parameters for amplification are as follows, initial denaturation at 94 ⁰C for 4 min, primer annealing at 54 ⁰C for 30 sec and extension at 72 ⁰C for 1.5 min. This step is repeated for 35 cycles in an automated DNA thermal cycler (Palm-Cycler Corbett Research, Australia). Final extension is done at 72 ⁰C for 10 min. The PCR products are run on 0.8 and 1.5 % agarose gel, and the gels stained with Ethidium bromide are visualized under the UV trans illuminator. Molecular size markers (lkb DNA Ladder, MBI Fermentas, Canada) are run concurrently. For colony PCR the template is prepared by picking up isolated colonies of bacteria using a sterile toothpick and suspending them in 10 μl of nuclease free water and boiling them at 99 ⁰C for 10 min in a PCR tube. The master mix containing other reagents of a PCR mixture is added later and the above mentioned program for amplification is followed.

EXAMPLE 6

The PCR detection is specific for Salmonella and differentiates between various typhoidal serovars and other common pathogenic and non-pathogenic bacteria.

To prove that the PCR based detection is specific for Salmonella, colony PCR with different Salmonella serovars and other common pathogenic and non-pathogenic bacteria listed in table 1, was carried out. The PCR is carried out as described above and the different sized amplification product when run on agarose gel are able to differentiate between the various serovars (The samples were loaded on the gel in the same order as in Table 1). PCR amplification in S. typhi samples produced two bands of 384 bp and 1043 bp, whereas S. paratyphi A produced only 384 bp band. All other bacteria are negative for both the products (Figure 2). Megablast analysis of the open reading frames (ORF) used for the assay has revealed that Salmonella enterica serovars Heidelberg and Newport will produce the 1043 bp band. Of the two serovars, Newport is used for the assay but it did not produce any band. The reason for this remains unknown. The specificity is tested again with the same set of samples but with higher template concentrations by taking 20 μl of stationary phase culture of all the bacteria, pelleted and resuspended in 10 μl of water as the PCR template. When the annealing temperature is reduced to lesser than 50 ⁰C, non specific bands appear. Hence, for all further experiments annealing temperature of 54 ⁰C is used. The processing of whole blood for template DNA results in host genomic DNA contamination which can serve as a template for nonspecific binding and therefore can give rise to spurious PCR products. To rule out this possibility, the PCR assay is repeated with dilutions done in blood instead of PBS. The results of this experiment are found to be similar with no spurious PCR products, though the intensity of the bands is lesser. This may be because of the huge amount of contaminating proteins which may reduce the efficiency of the PCR.

EXAMPLE 7

The genomic loci which is selected as a diagnostic marker is potentially stable: The genomic locus which is used as a diagnostic marker should be genetically stable. An unstable region poses the possibility of it being deleted in certain isolates. This excision can take place through prophage activation in the case of bacteriophage sequences and through specific recombinase in pathogenicity islands and by homologous recombination between paralogous genes in the same bacterium. The genetic stability of the amplified region is tested by amplifying the region from clinical isolates from various parts of India. Twenty samples were obtained from JEPMER, Pondicherry (South India) and 12 samples from Nagpur (Central India). The PCR assay conducted with pure cultures of these clinical isolates amplified the expected band from all the isolates (Figure 3).

Though the sample of 32 is less to rule out the presence of allelic variants of the gene or mutants with complete deletion of the gene, further proof for stability of that particular genomic locus is gathered by bioinformatics analysis. In silico analysis is carried out to confirm that this region is not in any mobile pathogenicity islands or bacteriophage insertions by analyzing the GC percentage and subjecting the entire region to BLAST analysis to look for regions of homology to bacteriophage genome.

EXAMPLE 8

The PCR based assay can detect as low as four bacteria per ml of blood: After determining the specificity and stability of the chosen diagnostic marker gene, the sensitivity of the assay is measured. The minimum number of bacteria that can be detected in one round of PCR amplification with 35 cycles is determined by using serial dilutions of pure culture of S. typhi as templates. O.D (600nm) of bacteria is set to 0.3 which contains 1.5 x 10⁸ bacteria/ml (Precalculated through dilution plating), from which serial dilution is made. The PCR was repeated 4 times and the lowest dilution which produced band consistently is taken as the lowest detection limit. The dilutions are carried out in both blood and PBS and in both the cases, bacteria as low as one in 300μl of blood, gave rise to the bands corresponding to the desired product size (Figure 4). Absolute sensitivity, in case of PCR, depends on the ability to amplify very low number of template DNA molecules, consistently, which in this case is the genomic DNA of bacteria. Detection limits can be increased by doing two rounds of PCR, by using the product of one round of amplification as the template for the next round but this increases the time and the cost. A major objective of this PCR diagnostic assay is to develop a reliable, easy, quick and economic means of diagnosis of typhoid fever in the developing countries. To reduce the total time and cost of the assay, the maximization of the detection level in a single round of PCR amplification without compromising on the specificity, is done. By taking 300 μl of blood as the template, detection of > 4 bacteria/ml of blood in a single round of PCR, is achieved. EXAMPLE 9

The PCR assay is more sensitive than WIDAL test: The conventional means of diagnosis of typhoid are the WIDAL test and blood culture test. The blood culture tests though time consuming is considered the gold standard in typhoid diagnosis. The sensitivity of the PCR assay with the given primer set is calculated as against the WIDAL test and blood culture test which are the conventional means of diagnosis for typhoid. Blood samples from patients suspected of typhoid fever, showing symptoms like pyrexia are collected before antibiotic treatment from Indian Institute of Science (IISc) health centre and the R.V diagnostic centre, Bangalore, India. Of these total samples, 58 samples are subjected to WIDAL test, PCR diagnosis and Blood culture test. 20 samples are subjected to PCR and WIDAL test (Table 2). A representative gel picture along with the WIDAL results shows that PCR assay is more sensitive than WIDAL test (Figure 5). Taking blood culture as the standard, WIDAL test has only 63 % sensitivity where as PCR method has 95 % sensitivity (Among 58 samples with culture result). Using the sample set for which data is available for all three diagnostic methods namely PCR, WIDAL and blood culture, chi-square analysis using 2 x 2 contingency tables are done to compare the performance of the three methods. The PCR detection method when compared with the blood culture method revealed no significant difference (P > 0.05). Whereas, there is significant difference between WIDAL test and blood culture (P < 0.05). Comparison between WIDAL test and PCR analysis shows that PCR test is significantly better than WIDAL (P < 0.05). Among the 20 samples for which culture assay is not done PCR assay detected 11 positive samples as compared to only 5 positive samples detected by WIDAL test. Table 2:

The uniqueness of the instant method and the added advantage over other methods, stems from 2 features of the present invention.

1. The direct processing of blood, to be used as PCR template makes it easier, quicker and cheaper. It differs from other inventions where DNA is purified from the blood using columns.

2. The uniqueness of the primer lies in the fact that it has higher specificity in terms of differentiating the typhoidal strains from the non-typhoidal strains and also that it can differentiate Salmonella typhi from Salmonella paratyphi which is an added advantage in determining the course of treatment.

CONCLUSION

The use of two novel sets of primers from two unique regions of S. typhi is a remarkable improvement upon the existing methods of PCR based diagnosis because simultaneous amplifications increases the specificity of the test similar to two rounds of PCR with nested primers. In previous studies, where a nested PCR reaction is carried out for the diagnosis of Salmonella, use of one or two sets of primers in the second round of PCR is done. Though the nested PCR increases detection limits by increasing the number of cycles of amplification, the increased sensitivity is equal to a single round of PCR with two sets of primers amplifying two different loci, which have been employed in the present study. The present disclosure depicts that the detection level of bacteria with a single round of amplification of 35 cycles in the present study is very high, where as low as one CFU is detected. Hence, the present method is able to achieve specificity similar to a nested PCR along with a high sensitivity in one round of amplification thus saving time and reagents. An estimated number of bacteria per ml of peripheral blood during the bacteremic phase in case of typhoid is one CFU/ml. As the present method is able to detect this range, it can be safely used for diagnosis without the fear of false negatives.

The primers used in this study are designed against a potentially stable genomic locus. Clinical isolates from different parts of India produced the expected band size. Most of the previous studies, used primers designed against the hypervariable region of the flagellar genes which are the regions susceptible to high mutation rates. The use of a hypervariable gene as a diagnostic marker precludes the possibility of, emergence of variants of the gene which can give a dangerous false negative result in PCR diagnosis using flagellar genes and serological tests involving detection of flagellar antibodies.

The use of whole blood lysate as PCR template is a modification over other previous studies involving the purification of DNA from blood which is a costly and time consuming process. In the present method, the use of whole blood lysate instead of purified DNA is done. This step reduces the need to purify DNA from blood which is an additional step and increases the cost and time. The present study makes use of 0.2% Triton-X 100 for lysis without any other buffer or lysis agent, this allows the use of lysate dissolved in this solution directly as template. Further, the PCR yield did not change when different commercial PCR enzymes and their cognate buffers are used. This gives the present invention the flexibility to use any commercially available PCR enzyme and buffer and eliminates the need for specialized buffer compositions. The present PCR test can also be used to determine the serovars of the Salmonella strain through a single PCR reaction of colonies isolated from blood culture, along with biochemical tests and slide agglutination tests. Serovar identification is crucial to predict the course of the disease and also the treatment regime as different serovars respond to different modes of treatment.

By using two pairs of oligonucleotides in a single PCR reaction instead of two reactions in nested PCR, the time taken for diagnosis is considerably reduced. The whole procedure to differentiate the typhoidal Salmonella in the blood by agarose gel electrophoresis takes only 5 h, demonstrating PCR to be rapid, specific and most reliable method for early diagnosis of typhoid fever.

The practical value of PCR in the clinical diagnosis is the detection of Salmonella DNA in the blood samples from patients with suspected clinical symptoms but with negative cultures. The low level of bacteraemia in typhoid patients can cause negative blood culture, particularly if the patients have been treated with antibiotics before culture. Depending on the conventional diagnostic methods, these culture negative cases cannot be confirmatively diagnosed as typhoid fever. Typhoid fever is a very debilitating disease and hence demands for very accurate and fast diagnosis. Pretreatment with antibiotics will lead to culture negative condition but will not kill the bacteria residing in gall bladder which will lead to carrier condition and relapse of fever. Even with antibiotic treatment, PCR diagnosis can detect dead bacteria in blood, thus helping to identify and monitor those patients who are susceptible for relapse of the disease or progress to a carrier state.

SEQUENCE LISTING

<110> INDIAN INSTITUTE OF SCIENCE

<120> PRIMERS, PCR REACTION MIXTURE AND METHODS THEREOF <130> IP12004

<150> 1448/CHE/2009

<151> 2009-06-18

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<170> Patentln version 3.5

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<213> Salmonella

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Claims

We claim:

1) Primers of SEQ ID Nos. 1, 2, 3 and 4. 2) The primers as claimed in claim 1, wherein the primers having SEQ ID Nos. 1 and 3 are forward primers and the primers having SEQ ID Nos.

2 and 4 are reverse primers and, the primers form a combination set forth as SEQ ID Nos. 1 and 2, and SEQ ID Nos. 3 and 4. 3) The primers as claimed in claim 1, wherein the primers having SEQ ID Nos. 1 and 2 correspond to genomic loci STY0312 in Salmonella typhi CT18 and SPA2476 in Salmonella paratyphi A respectively and, wherein SEQ JD Nos.

3 corresponds to STY0313 in Salmonella typhi CT18, SPA2475 in Salmonella paratyphi A and t2576 in Salmonella typhi Ty2 respectively and, wherein SEQ ID No. 4 corresponds to STY0316 in Salmonella typhi CT 18 and Salmonella typhi Ty2 respectively.

4) A PCR reaction mixture, said mixture comprising of Tris having a concentration preferably of about 67 mM and pH preferably of about 8.8, Ammonium Sulphate having a concentration preferably of about 16 mM, polyethylene glycol p-( 1,1,3,3- tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.1%, Magnesium Chloride having a concentration preferably of about 2.5 mM, dNTPs having a concentration preferably of about 200 μM, primers as set forth in SEQ ID Nos. 1, 2, 3 and 4, each having a concentration preferably of about 25 picomoles and Taq polymerase preferably about 2units.

5) A method of obtaining a template for PCR, said method comprising steps of:

(a) centrifuging blood sample to obtain a pellet;

(c) washing the centrifuged pellet with the polyethylene glycol p-(l,l,3,3- tetramethylbutyl)-phenyl ether; and

6) The method as claimed in claim 5, wherein the blood sample has a volume ranging from about 200 μl to about 300 μl, preferably about 200 μl, the centrifuging is carried out from about 10,000 g to about 12,000 g, preferably at about 12,000 g for a time duration of about 2 minutes to about 4 minutes, preferably about 2 minutes, the adding of polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.2% and a volume preferably of about 1.0 ml, followed by vortexing, incubating of the pellet at room temperature for a time duration ranging from about 5 minutes to about 10 minutes, preferably about 5 minutes, and re- washing of the pellet with the polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether, the suspending in nuclease-free water of volume ranging from about 15 μl to 30 μl, preferably of about 20 μl and the boiling temperature preferably at about 99 ⁰C for a time duration preferably of about 10 minutes.

7) A method of detecting Salmonella infection, said method comprising steps of:

(a) forming a PCR reaction mixture;

8) The method as claimed in claim 7, wherein the PCR reaction mixture comprises

Tris having a concentration preferably of about 67 mM and a pH preferably of about 8.8, Ammonium Sulphate having a concentration preferably of about 16 mM, polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.1%, Magnesium Chloride having a concentration preferably of about 2.5 mM, dNTPs having a concentration preferably of about 200 μM, the primers each having a concentration preferably of about 25 picomoles and Taq polymerase preferably about 2units. 9) The method as claimed in claim 7, wherein the primers having SEQ ID Nos. 1 and 3 are forward primers and the primers having SEQ ID Nos. 3 and 4 are reverse primers and, the sample is selected from a group comprising blood, serum and plasma. 10) A kit for detection of Salmonella, wherein said kit comprises primers of SEQ ID Nos. 1, 2, 3 and 4 along with the PCR reaction mixture.

11) The kit as claimed in claim 10, wherein the PCR reaction mixture comprises Tris having a concentration preferably of about 67 mM and a pH preferably of about

8.8, Ammonium Sulphate having a concentration preferably of about 16 mM, polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether having a concentration preferably of about 0.1%, Magnesium Chloride having a concentration preferably of about 2.5 mM, dNTPs having a concentration preferably of about 200 μM, the primers each having a concentration preferably of about 25 picomoles and Taq polymerase preferably about 2 units.