FORM 2
THE PATENTS ACT, 1970 (39 of 1970) &
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10, Rule 13]
PCR DIAGNOSTIC KIT AND METHODS FOR AMPLIFICATION AND DETECTION OF PATHOGENS;


GENESIS LABS LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS 45-47 M.G. ROAD, FAZALBHOY BUILDING, FORT, MUMBAI- 400 001, MAHARASHTRA, INDIA.

THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION.


Field of the Invention
The present invention relates to a diagnostic kit for detection of disease using Gene Sequence Amplification or Polymerase Chain Reaction technique.
Background
Polymerase chain reaction is the most sensitive of the Existing rapid methods to detect microbial pathogens in clinical specimens. In particular when specific pathogens that are difficult to culture in vitro or require a long cultivation period are expected to be present in specimens, the diagnostic value of PCR is known t0 be significant. However, the application of PCR to clinical specimens has limitations due to the susceptibility of PCR to inhibitors, contamination and experimental conditions.
Contamination is one of the major issues for rapid commercialization of this fast, sensitive and reliable technique for detection of ,pathqgens in dinicai samples. The PCR assay in diagnosis involves several critical steps, such as nucleic acid extraction from specimens, PCR amplification, and detection of ampliccos. During all these steps, the reaction tubes are exposed to air, to liquid handling devies and to instruments where post amplification analysis are done. During transfer of the amplified product from reaction tube to analysis system, the liquid handling devices create aerosolic particles containing the amplified nucleic acid, which not only enter the liquid handling devices, but also spreads into the air and stick to the skin and clothing of the personal handling the devices. Spread of aerosolic nucleic acid can be controllec| by using aerosol resistant tips for all PCR work. These are disposable tips that retai-d the entry of aerosolic nucleic acid in and out of the barrels of liquid handling device These tips are however very expensive and cannot form a barrier in case of carry over contamination.
If a new round of PCR tube for testing is set up in the same reaction area, or work area,
sharing the same enclosed airflow, or using the set of instruments used during post
amplification analysis, false positive results due to carry over contamination is obtained.
One way to remedy this is to prepare a three room setup wherein individual rooms are
dedicated for nucleic acid isolation, PCR-setup and post PCR analysis. Each room is
equipped with a separate airflow system and the work f|ow is always directed in the
following order: Nucleic acid isolation>PCR Setup>post PCR analysis and the order Is
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strictly unidirectional. However, for pathological laboratories to adopt GSA as routine diagnostic test for detecting infectious or genetic diseases, allotment of three dedicated rooms becomes constraining. Apart from dedicated rooms, the three steps also need dedicated equipment, especially liquid handling equipments which consist of both automatic as well as semi automatic devices. These equipments are extremely expensive and it's even more expensive to dedicate them to an individual type of work.
As aerosolic nucleic acid can be easily carried over through the clothes of the persons visiting the post PCR area, entry of people other than the technician performing post PCR analysis into the 3 rooms is restricted. This practice is again difficult to follow in small pathological labs where space is a constraint.
Accidental nucleic acid contamination is often treated with strong detergents and bleach. All work surfaces and equipments are thoroughly washed before and after each steps of PCR and spillage is strictly avoided. All possible equipments required for PCR are exposed to UV before and after performing the PCR. Clothes used in dedicated areas are either dedicated or washed immediately after use. Autoclaveable items are autoclaved. However, automatic and semi automatic devices which may have small nooks and corners tend to retain contamination. In an event of accidently spillage, discarding the equipments may be the only option left.
Thus, even after the implementation of the above remedies, PCR remains unpopular among pathological laboratories because of the huge cost involved in setting up dedicated rooms and equipment makes the test very expensive. Besides, accidental contamination of liquid handling devices which can never be removed may unknowingly provide false results. Additionally, the present format for polymerase chain reaction tests is too complicated to be adopted by a pathological lab technician hence specialized trained people are required to perform the test which again adds to the cost of polymerase chain reaction based detections particularly to patients in the need of such diagnostic tests.
Description of the invention
The problems described above have been overcome by providing a disposable
diagnostic Kit for Gene Sequence Amplification or Polymerase Chain Reaction based
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technique. The diagnostic kit provided by the present invention is in a disposable format and is suitable for use even in ordinary pathological laboratory environment by lab technicians.
In one embodiment of the present invention there is provided a disposable diagnostic kit for Gene Sequence Amplification or Polymerase Chain Reaction based technique comprising a plurality of tubes containing pre-aliquoted mix of PCR reagents and a plurality of sets of disposable detection tubes charecterised in that the PCR kit is provided with a plurality of sterile disposable micro dropper to replace automatic and semi-automatic liquid handling devices and eliminate carry over and aerosolic contamination.
Each kit as per the preferred embodiment of the present invention comprises a plurality of sets of one time use detection tubes containing a mix of primers nucleic acid for the pathogen to be detected and deoxyribonucleotide triphosphates hereinafter referred to as dNTP's for at least a single round of polymerase chain reaction, a plurality of sets of reagent tubes containing pre aliquoted mix of polymerase chain reaction reagents and a plurality of sterile disposable micro dropper for adding or mixing the reagent from the reagent tube to detection tube.
The detection tubes contain necessary and sufficient amount of reagents to perform at least one round of test. The vial along with its content is meant for disposal after use without leading to significant wastage.
In one of the embodiments the tubes of the PCR kit are color coded to facilitate the differentiation between different sets of detection tubes as well as reagent tubes and make the process much simpler and easy to understand for a routine laboratory technician. It further helps them to minimize errors and thus prevent false results.
In further embodiments of the present invention, the PCR kit is optionally provided with a nucleic acid extraction kit comprising of pre-aliquoted mix of nucleic acid extraction reagents in a plurality of separate color coded and numbered bottles and a plurality of disposable sterile droppers to avoid use of automatic and semi automatic liquid handling devices.
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The format of the disposable diagnostic kit of the present invention is simple and can be used in any ordinary pathological or research laboratory environment and even in field. The further advantage of the disposable detection kit of the present invention is that for the use of such kit no special training is required and can be even handled by pathologists in a simple manner similar to other simple diagnostic tests routinely carried out in pathological laboratory.
The PCR kit can be adapted for detection of various microbial and viral diseases such as HIV, hepatitis, TB, malaria etc by using the nucleic acid primers suited for the specific purpose. Multiple species detection in one go is also possible using the kit of the present invention without the need and use of sophisticated instruments as well as automatic and semiautomatic liquid handling devices. Possible applications of the kit also include genotyping studies, clinical and industrial research & development studies as well as a number of other routine PCR techniques.
In another embodiment of the present invention there is provided a method for performing nucleic acid sequence amplification and detection using the disposable diagnostic kit comprising the steps of;
a) extracting nucleic acid from the fluid sample acquired from the patient using standard nucleic acid extraction reagents in the kit;
b) adding a drop of pre-aliquoted reagent mix in the detection tube using micro dropper;
c) adding the extracted nucleic acid to detection tubes using a sterile micro dropper;
d) placing the detection tubes in a thermal cycler for amplification; and
e) analyzing the contents of detection tube for amplified nucliec acid.
It is evident from the above description that the disposable diagnostic kit of the present invention requires use of minimum number of equipments and devices to perform the polymerase chain reaction test. Use of disposable droppers and one time use detection tubes eliminates the chances of any carry over contamination to minimize false results.
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As per one of the preferred embodiment a PCR kit for detection of malaria is provided which comprises a set of three detection tubes color coded blue, yellow and white for detection of vivax, falciparum and Plasmodium species of malaria respectively. Each detection tube comprises a pre-aliquoted mix of PCR buffer, along with Taq DNA polymerase, MgCb and the oligonucleotide primer of the pathogen species to be detected. Optionally the kit is also provided with a set of two tubes color coded green and red for blank and control respectively. The blank and control tube comprise of Plasmodium primer along with PCR buffer, MgCI2 and Taq DNA polymerase.
A set of three reagent tubes i.e. reagent A, reagent B and reagent C and color coded purple, green and red are provided the contents of which are as follows:
Reagent A:- dNTP
Reagent B:- PCR grade water
Reagent C:- Cloned Plasmodium DNA
Optionally, the kit is further provided with a DNA extraction kit the contents of which are as follows:

Colour Coding Composition
DNA isolation reagent 1 in dropper bottles with markings at 0.2 and 0.5 ml Green Tris-HCl buffer, guanidine hydrochloride, Triton-X-100 and EDTA
DNA isolation reagent 2 in dropper bottles with markings at 0.2 and 0.5
ml Red Tris-HCl buffer pH 8.0 and NaCI, Alcohol
DNA isolation reagent 3 in dropper bottles with markings at 0.2 and 0.5 ml Yellow Tris-HCl buffer pH 8.0 and EDTA
DNA isolation reagent 4 Blue Absolute Alcohol
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in dropper bottles with markings at 0.2 and 0.5 ml
DNA purification columns with flow through collection tube Silica matrix columns with 2 ml collection tubes
Column wash tube 2ml
microcentrifuge tube
DNA elution tubes 2ml
microcentrifuge
tube
Droppers for DNA
isolation 1ml droppers
DNA isolation blood lysis tubes 1.5 ml
microcentrifuge
tube 20 microliter of 20mg/ml Proteinase K
Detection of pathogen starts with extraction of DNA from the blood sample. The DNA extraction steps using the kit of present of present invention can be summarized as follows:
a. Add approximately 0.2 ml of blood to the lysis tube
b. Add a drop of reagent 1 to the lysis tube, vortex for approximately 15 sees and
incubate at 55 °C
c. Add a drop of alcohol and vortex for approximately 15secs.
d. Load the lysed blood from the lysis tube into the DNA purification columns using
a sterile dropper
e. Centrifuge the column for 1 min at 6500 Xg and discard the flow through
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f. Wash the column with reagent 2 and again centrifuge for 1 min at 6500Xg and
discard the flow through
g. Repeat the step f again, performing centrifugation at top speed for 3 mins and
discard the flow through with the tube
h. Add a drop of reagent 3 into the column and incubate at room temperature for 5 mins
i. Centrifuge for 1 mins at 6500Xg, save the eluted DNA with the tube and discard the column
To perform the detection, 1 drop of reagent A is added using a fresh sterile micro-dropper followed by 1 drop of eluted DNA, added to each of the detection tubes using the micro-dropper. One drop of reagent B is added to the blank tube and one drop of reagent C is added to the control tube. All additions are done using individual fresh and sterile micro droppers, under hoods fitted with UV lamps and exhaust fans to avoid contamination. The tubes are then shut and are subjected to polymerase chain reaction using a thermocyler. The tubes are then analysed for the presence or absence of pathogen.
Dated this 17th day of October, 2008.
FOR GENESIS LABS LIMITED leir Agenl
(ALIASG^fc pHOLKAWALA) KRISHNA4 SAURASTRI
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