| Title of Invention | "PRIMERS FOR GENE AMPLIFICATION" |
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| Abstract | The invention relates to gene amplification method for in vitro detection of viral genome in a sample comprising designing primers for gene amplification; RNA extraction by heat release method; gene amplification by one-step single-tube RT-LAMP method; detection of gene amplification by naked eye viualisation. |
| Full Text | GENE AMPLMCAHON METHOD FOR EARLY DETECTION OF CfflKlUNGUNYA VIRUS FIELD OF INVENTION The present invention relates to the field of gene amplification. The present invention relates particularly to the development of a simple, rapid and low-cost method of gene amplification for early detection of Chikungunya virus genomic RNA in human patient serum samples. BACKGROUND AND PRIOR ART OF THE INVENTION Chikungunya virus (CHIKV), a member of the Alpha virus genus, is of considerable public health importance in Southeast Asian and African countries; India has also witnessed an explosive outbreak of unprecedented magnitude last year that has swapped across the country from South to North. This is one of the largest and most severe epidemic in the history of Chikungunya and it has resurged after a gap of 32 years affecting more than 10,00,000 persons covering almost all the states of India. The CHIKV produces an illness in human that is often characterized by sudden onset of fever, headache, fatigue, nausea, vomiting, rashes, myalgia and severe arthralgia. The arthralgia may persist in a small proportion of cases even for months and has major economic impact in many tropical countries. The clinical symptoms of Chikungunya mimic those of dengue fever and therefore, many cases of chikungunya are misdiagnosed as dengue infections. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, only a few specific serological or molecular diagnosis tools are available. CHIKV diagnosis is based essentially on virus isolation, RT-PCR and ELISA assays. These assays are described in prior art for specific detection of CHIKV in mosquitoes for clinical samples. The conventional RT-PCR methods have been reported for the study of CHIKV replication in supernatants, clinical samples or for epidemiological survey. In spite of high magnitude of amplification, these PCR based methods require either high precision instruments for the amplification or elaborate methods for detection of the amplified products. In addition, these methods also suffer from low sensitivity thereby missing border line cases especially on early stage of the disease and are often cumbersome to adapt for routine clinical use especially in peripheral health care settings and private clinics. Therefore, a rapid, specific and sensitive test is necessary for effective surveillance of new CHIKV circulating strains. More recently, Pastorino et. al, 2005 reported a fully automatic Taq-man real-time RT-PCR assay for the detection of CHIK virus in acute-phase serum samples. The real-time PCR assay has many advantages over conventional RT-PCR methods, including rapidity, quantitative measurement, lower contamination rate, higher sensitivity, higher specificity, and easy standardization. Need for the Present Invention All these nucleic acid amplification methods have several intrinsic disadvantages of requiring either a high precision instrument for amplification or an elaborate complicated method for detection of amplified products. Therefore, these rapid molecular tests might not be the method of choice in basic clinical setting in developing countries or in field situations because of the requirement of sophisticated instrumentation and expensive reagents. It is therefore critical to develop simple and economical molecular tests that can be used in field conditions especially at peripheral healthcare setting as a routine test without requirement of any sophisticated high end equipments. OBJECTS OF THE INVENTION It is an important object of the present invention to develop a field based gene amplification method that can be used in a routine diagnostic laboratory as well as in peripheral health care centers for early detection of Chikungunya virus in acute phase human patient serum samples. Another object of the present invention is to design a highly sensitive and specific primer set to identify and pick up very low copy number of the Chikungunya virus genome with high degree of specificity enabling early differential diagnosis from other viruses having similar clinical spectrum such as Dengue virus transmitted by the same mosquito Aedes aegypti. Yet another object of the present invention is to develop a rapid and reliable method of detection and identification of Chikungunya virus at an early stage of disease. Still another object of the present invention is to develop a simple and inexpensive method of detection and identification of Chikungunya virus without requiring any sophisticated detection equipments at any stage. An additional object of the present invention is to develop a simple method of sample processing (RNA extraction) without requiring expensive kits. Further object of the present invention is to develop a quick method of sample processing (RNA extraction) without compromising with sensitivity and specificity of the assay system. SUMMARY OF THE INVENTION Accordingly, the present invention provides for a method for in vitro detection of viral genome in a sample comprising the steps of: designing primers for gene amplification; extracting RNA by heat release method; amplifying gene by one-step single-tube RT-LAMP method, wherein the said RT- LAMP method comprises: preparing a reaction mixture of primers, buffer, DNA polymerase, Reverse Transcriptase and sample/RNA template in a single tube; subjecting the reaction mixture to incubation at 63°C for a duration of at least 30 min to give a RT-LAMP product; detecting amplified gene in the said RT-LAMP product. In one embodiment of the method of the invention further comprises validation of the said sample. In another embodiment of the method of the invention the said sample is patient serum sample. In a preferred embodiment of the method of the invention the primers are a set of six primers comprising two outer, two inner and two loop primers; wherein the said primers have the following sequence: Forward Outer (F3) of SEQ ID No. 1 ; Backward Outer (B3) of SEQ ID No. 2; Forward Inner Primer (FIP = [Flc+TTTT+F2]) of SEQ ID No.3+TTTT+SEQ ID No.4; Backward Inner Primer (BIP=[Bl+TTTT+B2c] of SEQ ID No.5+TTTT+SEQ ID No.6; Forward Loop Primer (FLP) of SEQ ID No. 7; Backward Loop Primer (BLP) of SEQ ID No. 8. In a further embodiment of the method of the invention the RNA extraction is accomplished by heating the sample in the temperature range of 50°-90° C for a duration in the range of 10-30 min; wherein the RNA extraction is preferably accomplished by heating the sample at 60°C for a duration of at least 10 min. In a yet another preferred embodiment of the method of the invention the detection of gene amplification in the said RT-LAMP product is accomplished by naked eye visualization of turbidity. In a still another preferred embodiment of the method of the invention a fluorescent dye is added to the said RT-LAMP product for the detection of gene amplification by naked eye visualization of fluorescent dye mediated colour change and/or visualization by illumination under UV-lamp; wherein the said dye is preferably SYBR Green I dye. DETAILED DESCRIPTION OF THE INVENTION LAMP which stands for Loop-Mediated Isothermal Amplification is a novel nucleic acid amplification method developed by Eiken Chemical Co., Ltd Japan and has the potential to replace PCR because of its simplicity, rapidity, specificity and cost-effectiveness (Notomi et. AI.2000). The RT-LAMP assay has emerged as a powerful gene amplification tool for rapid identification of microbial infections and is being used for rapid detection and typing of emerging viruses viz. West Nile (WN), Severe Acute Respiratory Syndrome (SARS), Dengue (DEN), Japanese Encephalitis (JE) etc. in our laboratory (Parida et. al.2004; 2005; 2006 & 2007). The RT-LAMP assay is a simple diagnostic tool in which the reaction is carried out in a single tube by mixing of the buffer, primers, reverse transcriptase and DNA polymerase, and incubating the mixture at 63°C for 30 minute. The higher sensitivity and specificity of the RT-LAMP reaction is attributed to continuous amplification under isothermal condition employing six primers recognizing eight distinct regions of the target. Besides, the higher amplification efficiency of RT-LAMP reaction yields large amount of by-product, pyrophosphate ion, leading to white precipitate of magnesium pyrophosphate in the reaction mixture. Since the increase in turbidity of the reaction mixture according to the product of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the RT-LAMP reaction can be achieved by real-time measurement of turbidity. The execution of the RT-LAMP reaction and the measurement of its turbidity are extremely simple as compared to the existing real-time Taqman TR-PCT and NASBA assays that require fluorogenic primers and probes as well as expensive detection equipments. One of the most attractive features of RT-LAMP assay seems to have great advantage in terms of monitoring of amplification that can be accomplished by SYBR Green I dye mediated naked eye visualization and by real time monitoring by using an inexpensive turbidimeter according to the situation. Still the requirement of turbid meter which costs around $20,000 is another constraint for use of RT-LAMP in field conditions. The present invention describes the development of a heat inactivated SYBR Green I based one step single tube accelerated RT-LAMP assay for rapid and real-time detection of CHIK virus in clinical specimens by targeting the immunodominant structural El gene that has potential usefulness in clinical diagnosis as well as surveillance of Chikungunya virus in developing countries like India. Of particular importance is the substantial reduction in time required for the confirmation of results by RT-LAMP assay i.e. in 30 min as compared to 3-4 hours in case of RT-PCT as well as no requirement of RNA extraction kit and expensive sophisticated detection equipment thereby making it very simple, rapid and low cost gene detection technology with tremendous clinical diagnosis potential. Process Steps: The simple, rapid and low-cost gene amplification method of the present invention for the early detection of Chikungunya virus genomic RNA in human patient serum samples can be summarized as follows: Step 1: Primer designing Step 2: RNA extraction Step 3: Gene amplification Step 4: Naked Eye Visualization of Gene amplification Step 5: Validation of Patient serum samples Step 1 (Primer designing) A set of six primer comprising two out, two inner and two loop primers that recognize eight distinct regions spanning over 205 bp conserved regions of structural El gene corresponding to the genome position 10,294 to 10,498 was designed employing the LAMP primer designing support software program (Net laboratory, Japan, http://venus.netlaboratory.com). The two outer primers were described as forward outer primer (F3) of SEQ ID No. 1 and backward outer primer (B3) of SEQ ID No. 2. The inner primers are described as forward inner primer (FIP) and backward inner primer (BIP). Further, two loop primers viz; forward loop primer (FLP) of SEQ ID No. 7 and backward loop primer (BLP) of SEQ ID No. 8 were designed to accelerate the amplification reaction. FIP consists of a complementary sequence of Flc (SEQ ID No. 3) and a sense sequence of F2 (SEQ ID No. 4). BIP consists of a complementary sequence of Bl (SEQ ID No. 5) and a sense sequence of B2c (SEQ ID No. 6). FIP and BIP were high performance liquid chromatography (HPLC) purified primers. The FLP and BLP primers were composed of the sequences that are complementary to the sequence between Fl and F2; Bl and B2 regions respectively. The usefulness of the selected primer set in correctly identifying all genotypes of CHIKV was established through sequence alignment of all available El gene sequences in the Genbank including the circulating strains in India responsible for recent epidemics using DNASIS software. The details of the primer sequences with regard to genome position are depicted in the Table-1. Step 2 CRNA extraction by Heat Release Method) In an attempt to minimize the sample processing time as well as cost, a series of experiments were carried out to find out the most effective method of virus RNA release without employing expensive kits and time consuming tedious multiextraction steps. The extraction of genomic viral RNA was achieved by adapting a very simple method of heat release though boiling of samples at different temperature (50-90°C) for different durations (10-30 min). The destabilization of the physical integrity of viruses by heating resulted in the release of viral RNA which is then potentially available for detection by RT-LAMP. However, the temperature at which virus particles become disintegrated during heating differed significantly between virus types and physicochemical conditions. The optimum temperature and shortest incubation period that can give best results without compromising with sensitivity and specificity was found to be 60°C for 10 min. This has not only saved the time but also obviates the requirement of very expensive QIAmp viral RNA mini kit (QIAGEN, Germany) kit (Costing Rs. 250/sample) routinely used in the laboratory by researchers all over the world. Step 3 (Gene amplification by one-step single-tube method^ The RT-LAMP reaction was carried out in a total 25 ul reaction volume using the in-house buffer comprising 20mM Tris-HCl pH8.8, lOmM KC1, lOmM (NH^SO,,, 8mM MgSO4, 1.4 mM dNTPs, 0.8M Betaine, 0.1% Tween-20 with 50 pm9ol each of the primers FIP and BIP, 5 pmol each of the outer primers F3 and B3, 25 umol each of loop primers FLP and BLP, 8 units of the Bst DNA polymerase (New England Biolabs), 0.625 units of the AMV Reverse Transcriptase (Invitrogen) and 2 ul of RNA template. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 30 min in routine laboratory water bath. This one-step single-tube method was employed in order to amplify the viral RNA in a simple and cost-effective manner. The method is inventive owing to simultaneous addition of reverse transcription and polymerization thereby standardizing a one-step single-tube method for RNA amplification. This coupling of reverse transcription and polymerization not only reduces the time but is also substantially cost-effective over prior art methods. Here it must be noted that RNA is not directly amenable to be amplified by the commonly used DNA Polymerase. The application of the said one-step single-tube method of RNA amplification can be extended to other genetic material e.g. DNA also, and such a modification of this method is within the scope of the invention. Step 4 (Naked Eye Visualization of Gene amplification) In order to facilitate the field application of RT-LAMP assay, the monitoring of RT-LAMP amplification was also carried out with naked eye inspection. Following amplification, the tubes were inspected for white turbidity through naked eye after a pulse spin to deposit the precipitate in the bottom of the tube. The inspection for amplification was also performed through observation of color change following addition of 0.5 ul of SYBR Green 1 dye to the tube. In case of positive amplification, the original orange color of the dye will change into green that can be judged under natural light as well as under UV light (302nm) with the help of a hand held UV torch lamp. In case there is no amplification, the original orange color of the dye will be retained: This change of color is permanent and thus can be kept for record purpose. SYBR Green-I florescent dye mediated monitoring of CHIKV RT-LAMP amplification is illustrated in Figure-1. Step 5 (validation of Patient serum samples) The applicability of this modified SYBR Green 1 based heat inactivated RT-LAMP assay for clinical diagnosis was validated with acute phase human patient serum samples of the 2006 epidemic of CHIKV in Andhra Pradesh as well as the most recent 2007 Kerala epidemic. The said samples were collected from patients and tested under field-like conditions in strict secrecy environment of Laboratory. The results were compared with conventional RT-PCR. The comparative evaluation of this field based SYBR Green I based RT-LAMP assay with more than 150 randomly selected clinical samples revealed a very good concordance of 93% with that of RT-PCR. This modified field based RT-LAMP approach has demonstrated higher sensitivity by correctly picking up additional positive samples with low level of virus that were missed by RT-PCR. Comparative analysis of Heat coupled SYBR-Green I based RT LAMP and conventional RT-PCR assay for the detection of CHIKV in 135 suspected acute phase serum samples are presented in Table-2. It is noteworthy that none of the RT-PCR positive cases were missed by RT-LAMP process of the invention. In addition to RT-PCR positive cases, RT-LAMP picked up few additional (19) positive borderline cases with low copy number of viral RNA below the detection limit and thus missed by RT-PCR. Further retrospective analysis of these RT-LAMP positive samples revealed that these are true positive samples indicated by the presence of virus specific IgM antibodies and/or successful virus isolation. So these may be the borderline cases with very low level of viremia collected during the window period of the disease, where the viremia is in decline (undetectable by RT-PCR) and IgM antibody started appearing to a detectable limit. Furthermore, the assay protocol has been extensively validated with large number of clinical samples at NIMS, Hyderabad through third party evaluation under confidentiality agreement. MAIN ADVANTAGES OF THE INVENTION: 1. The invention provides a field based gene amplification method that can be used in a routine diagnostic laboratory as well as in peripheral health care centers. 2. The method provided by the invention is very simple and obviates the need of expensive high end equipment such as PCR machine, Gel Documentation system and commercial RNA extraction kit. 3. The RNA extraction was accomplished by Heat Release Method i.e., by simply heating the sample at 60°C for 10 min. 4. One-step single-tube method was employed in order to amplify the viral RNA and the gene amplification can be accomplished directly with 2 ul of heat inactivated serum samples in an ordinary water bath or dry heating block at 63°C for 30 min. 5. The result can be monitored either in the form of turbidity or change of colour through naked eye. The better appreciated result of apple green fluorescence can be achieved by a simple UV hand held torch. 6. The test is very rapid where the result can be obtained in 40 min as compared to 5-6 hour by conventional gene amplification assays. 7. The method is high sensitive and specific that can detect very low copy number of viruses especially in those borderline cases that may be missed by conventional RT-PCR techniques. SEQUENCE LISTING of the primers designed are as follows: THE DIRECTOR GENERAL, DEFENCEC RESEARCH AND DEVELOPEMENT ORGANISATION PARIDA, Manmohan SANNARANGIAH, Santhosh TRIPATHI, Nagesh Kumar DASH, Paban Kumar RAO, Putcha Venkata Lakshmana Gene Amplification Method For Early Detection of Chikungunya Virus SNP-4187, DG-DRDO 8 Patentlnversion3.3 1 19 DNA Artificial Artificially designed and synthesised Forward Outer Primer (F3) 1 acgcaattga gcgaagcac 19 2 19 DNA Artificial Artificially designed and synthesised Backward Outer Primer (B3) 2 ctgaagacat tggccccac 19 3 22 DNA Artificial Artificially designed and synthesised oligonucleotide (Flc) 3 cggatgcggt atgagccctg ta 22 4 20 DNA Artificial Artificially designed and synthesised oligonucleotide (F2) 4 tggagaagtc cgaatcatgc 20 5 23 DNA Artificial Artificially designed and synthesised oligonucleotide (Bl) 5 tccgcgtcct ttaccaagga aat 23 6 20 DNA Artificial Artificially designed and synthesised oligonucleotide (B2c) 6 ttggcgtcct taactgtgac 20 7 17 DNA Artificial Artificially designed and synthesised Forward Loop Primer [FLP] 7 gctgatgcaa attctgt 17 8 17 DNA Artificial Artificially designed and synthesised Backward Loop Primer [BLP] 8 cctatgcaaa cggcgac 17 We claim: 1. A method for in vitro detection of viral genome in a sample comprising the steps of: (i) designing primers for gene amplification; (ii) extracting RNA by heat release method; (iii) amplifying gene by one-step single-tube RT-LAMP method, wherein the said RT-LAMP method comprises: preparing a reaction mixture of primers, buffer, DNA polymerase, Reverse Transcriptase and sample/RNA template in a single tube; subjecting the reaction mixture to incubation at 63°C for a duration of at least 30 min to give a RT-LAMP product; (iv) detecting amplified gene in the said RT-LAMP product. 2. The method as claimed in claim 1 further comprising validation of the said sample. 3. The method as claimed in claims 1 and/or 2, wherein the said sample is patient serum sample. 4. The method as claimed in any preceding claim, wherein the said primers are a set of six primers comprising two outer, two inner and two loop primers. 5. The method as claimed in claim 4, wherein the said primers have the following sequence: Forward Outer (F3) of SEQ ID No. 1; Backward Outer (B3) of SEQ ID No. 2; Forward Inner Primer (FIP = [Flc+TTTT+F2]) of SEQ ID No.3+TTTT+SEQ ID No.4; Backward Inner Primer (BIP=[Bl+TTTT+B2c] of SEQ ID No.5+TTTT+SEQ ID No.6; Forward Loop Primer (FLP) of SEQ ID No. 7; Backward Loop Primer (BLP) of SEQ ID No. 8. 6. The method as claimed in any preceding claim, wherein the RNA extraction is accomplished by heating the sample in the temperature range of 50°-90° C for a duration in the range of 10-30 min. 7. The method as claimed in claim 6, wherein the RNA extraction is preferably accomplished by heating the sample at 60°C for a duration of at least 10 min. 8. The method as claimed in any preceding claim, wherein the detection of gene amplification in the said RT-LAMP product is accomplished by naked eye visualization of turbidity. 9. The method as claimed in any preceding claim, wherein a fluorescent dye is added to the said RT-LAMP product for the detection of gene amplification by naked eye visualization of fluorescent dye mediated colour change and/or visualization by illumination under UV-lamp. 10. The method as claimed in claims 8 and/or 9, wherein the dye is SYBR Green I dye. 11. A gene amplification method for in vitro detection of viral genome in a sample such as substantially hereinbefore described and with reference to the foregoing examples and accompanying drawings. |
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| Patent Number | 270217 | ||||||||||||||||||
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| Indian Patent Application Number | 625/DEL/2008 | ||||||||||||||||||
| PG Journal Number | 49/2015 | ||||||||||||||||||
| Publication Date | 04-Dec-2015 | ||||||||||||||||||
| Grant Date | 02-Dec-2015 | ||||||||||||||||||
| Date of Filing | 12-Mar-2008 | ||||||||||||||||||
| Name of Patentee | DEFENCE RESEARCH AND DEVELOPMENT ORGANIZATION | ||||||||||||||||||
| Applicant Address | MINISTRY OF DEFENCE, GOVERNMENT OF INDIA, ROOM NO.348, B-WING, DRDO BHAWAN, RAJAJI MARG, NEW DELHI-110 011,INDIA | ||||||||||||||||||
Inventors:
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| PCT International Classification Number | C12Q1/68; C12P19/34 | ||||||||||||||||||
| PCT International Application Number | N/A | ||||||||||||||||||
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