FIELD OF INVENTION
This invention relates to a multiplex PCR-based process for detecting Chlamydia trachomatis and Neisseria gonorrhoeae.
BACKGROUND OF INVENTION
In India Chlamydia trachomatis and Neisseria gonorrhoeae, are detected by separate method in spite of the fact about 50% of the sample are co-infected. The usual method of detection is Gram-staining followed by confirmation like, antigen detection or biochemical assay. Both these methods are highly unsatisfactory, especially in asymptomatic patient (where the infection load is low). At present in India few private pathological laboratory are carrying out PCR based diagnostic, for which they are completely dependent on the import of kit. There is no indigenous diagnostic kit available for Chlamydia trachomatis and Neisseria gonorrhoeae at present.
The drawbacks of the term existing state of art is as follows:
Culture method: Sensitivity of this method is as 50% as organisms may lose infectivity during transportation and storage, which will reduce the likelihood of propagation. In addition, the surface area of the cell culture influence the sensitivity. Cell culture, however, is time consuming, laborious and expensive and can therefore be provided by only a few central laboratories.

Antigen Detection: The diagnostic efficacy of these methods is not high enough to warrant clinical use unless the need for a fast result overweighs the lower diagnostic accuracy. Also, the ELISA tests may reveal positive results in the presence of other organisms such as E. coli and Bacteroides sp, and Staphylococcus aureus may be captured instead of Chlamydia due to binding to the Fc region of the antibodies, thereby causing false-positive reactions. DFA requires skilled personnel in order to differentiate C. trachomatis organisms from non-specific fluorescent particles.
DNA/RNA Detection: The diagnostic performance of non-amplified probe technique is not substantially different from that of the best ELISA.
Nucleic acid amplification tests (NAATs): Target gene for NAATs The Plasmid: Some studies give evidence or suggest that the plasmid-free variants are present in clinical samples, and although it may seem that plasmid is involved in DNA replication, it has been possible to culture a plasmid-free variant. Thus, the infections caused by plasmid-free variants will be undetected if the plasmid is used as target gene.
The 16S-rRNA gene: Due to high homology of the 16S rRNA gene with other organisms, optimal reaction conditions are crucial in order to avoid annealing of primers to 16S-rRNA genes of the other organisms that are present in all non-sterile clinical samples.
a) Inventor's method is based on choosing unique of the genome while in other commercial kit it is cryptic plasmid. The disadvantage of this


method is that plasmid is not present in all the serotypes/clinical isolates (Diane et.al. Infection and Immunity, December 1998, p. 6010-6013, Vol. 66, No. 12) The plasmid from the new Swedish variant has a 377 bp deletion in the first predicted coding sequence. Also found 44 bp duplication downstream of the deletion resulting in negative diagnosis. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. (Helena et. al. BMC Genomics 2009, 10:239 doi: 10.1186/1471-2164-10-239, Unemo M, Microbiology. 2010 Jan 21. [Epub ahead of print]. Our kit also provides a new transport medium that is not only suitable for collecting the sample but also storing the sample at 4°C for several days. An internal control has also been designed against human gene to test for any PCR inhibition in the sample.
b) Inventors have taken our gene as target since it is genomic DNA we are targeting, it is very stable. In the kit of Mahony et. al. 16S ribosomal RNA gene was used (Mahony et. al. JCM, 95 Nov; 33(ll):3049-53, Mahony et. al. 200lApr;(4): 1429-35), since RNA is highly labile and hence the sample quality and storage conditions became critical in getting positive results.
c) Both Roche commercial kit as well as kit proposed by Mahony et.al required a long method of detection of the PCR product (e.g. Roche required 24 hours while kit developed by Mahony et.al required either southern blotting or Eleectro chemilumnisense). Both of these method take several hours (4-24hours) to develop and are very expensive, in contrast our method is highly simple and one can visualize the amplicon on Agarose gel within 30 minutes-lhr.


OBJECTS OF THE INVENTION
An object of this invention is to propose a PCR-based prototype kit for detecting Chlamydia trachomiatis and Nesseria ganorrhoeae simultaneously and individually.
Another object of this invention is to propose a kit which is cost effective.
Further object of this invention is to propose a kit which reduces a kit which reduces the chances of error and any cross contamination.
Still further object of this invention is to propose a kit which can be operated without any technical expertise.
STATEMENT OF THE INVENTION
This invention relates to A multiplex PCR based process for detecting Chalamydia trachomatis and Neisseria gonorrhoeae, using, i) a reaction mixture having primers, wherein the primers are
Primers sequences of Phospholipase D Endonuclease Superfamily gene , used in diagnosis of Chlamydia trachomatis generates 368-bp product.
Primers sequence used for detection of C. trachomatis
(Sequence Removed)
[SEQ ID No.l]
(Sequence Removed)
[SEQ ID No.2


Alternate primers
5'-AACCCACTTCTTCCACAGTTTCTTCTAA-3' [SEQ ID No.3] S'-(Sequence Removed)
[SEQ ID No.4]
Primers sequences of Orfl gene used in diagnosis of Neisseria gonorrhoeae, generates 260-bp product.
Primers sequence used for detection of N. gonorrhoaea 5,-(Sequence Removed)
[SEQ ID No.5J 5'- (Sequence Removed)
[SEQ ID No.6]
Alternate primers
(Sequence Removed)
[SEQ ID No.7] (Sequence Removed)
[SEQ ID No.8]
Internal control Primers sequences used in prototype kit as an internal control.
Primers sequence used for internal control
(Sequence Removed)
[SEQ ID No.9] (Sequence Removed)
[SEQ ID No. 10]
Alternate sequences
(Sequence Removed)
[SEQ ID No. 11] (Sequence Removed)
[SEQ ID No. 12]


ii) a transport medium of 130 mM Nacl, 2.6mM KCI, lOmM Na2 HPO4, 1.7 mM KH2P04, lOmM EDTA, (for sample collection) iii) solution A of Tris (50 mM), EDTA (ImM) Tritonx 100 (1%) and solution B of proteinase K (200 ug/ml) for sample processing Wherein the steps comprising, collection of sample in one ml of transport medium, preparation of sample DNA by taking 500 of collected sample cocktail, centrifuging at 15, 000 g for 30 minute at 4°C, discarding the supernatant carefully without disturbing the pellet, mixing appropriate amount of solution A 48µl +solution B 2µl, suspending the pellet in 50 µl of above solution, incubating at 37°C for 1 hr, incubating the sample at 100 °C for 10 min, Spinning at 10, 000 rpm for 2 min and collecting the supernatant carefully, Setting up PCR with 8µl sample DNA, 26)µl reaction mixture 1 and 16µl reaction mixture II, wherein the PCR condition is one cycle of 94 °C for 5 minutes followed by 35 cycles of step 2 to step 4 at 95 °C, 63 °C and 72 °C for 30 seconds each and finally one cycle of 72 °C for 5 min.
DETAILED DESCRIPTION OF THE INVENTION
In the present invention a multiplex PCR based prototype kit for the detection of the Chlamydia trachomatis and Neisseria gonorrhoeae, based on designed primers (patent in process). The kit contains all the reagents for collection of clinical samples, for carrying out amplification by PCR and the detection of the product, as well as the protocol to be used for diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae in patient samples. Furthermore, three kits have been developed.


Process for diagnosis of samples infected with Chlamydia trachomatis with an internal control.
Process for diagnosis of sample infected with Chlamydia trachomatis without internal control.
Process for detection of Chlamydia trachomatis and Neisseria gonorrhoeae simultaneously.
Sample is collected in one ml of transport medium (130 mM NaCI, 2.6 m M KCI, lOmM Na2HP04, 1.7 mM KH2P04, lOmM EDTA).
Preparation of Sample DNA: 1. Take 500 µl of collected sample cocktail, in a fresh microentrifuge tube (1.5ml) and centrifuge at 15,000 g, for 30 minute at 4.degree. C.
2. Discard the supernatant carefully without disturbing the pellet.
3. Mixed the appropriate amount of solution (Solution A, 48.
mu. 1+Soluiton B, 2 . mu.l) before use.
3. Solution A consists of Tris (50 mM), EDTA (ImM) and Triton, times. 100
(1%) and solution B is proteinase k (200 µl/ml).
4. Suspend the pellet from step 3 in 50 p.11 of the above solution.
5. Incubated at 37. degree.C. for 1 hr.


6. Now incubate the sample at 100. degree. C. for 10 minute.
7. Spin at 10,000 rpm for 2 minute.
8. Collect the supernatant carefully in separate tube (Designated as
Sample DNA).
Primers sequences of Phospholipase D Endonuclease Superfamily gene , used in diagnosis of Chlamydia trachomatis generates 368-bp product.
Primers sequence used for detection of C. trachomatis
(Sequence Removed)
[SEQ ID No.l] (Sequence Removed)
[SEQ ID No.2]
Alternate primers
(Sequence Removed)
[SEQ ID No.3](Sequence Removed)
[SEQ ID No.4
Amplified region of gene used in diagnosis
(Sequence Removed)
Primers sequences of Orfl gene used in diagnosis of Neisseria gonorrhoeae, generates 260-bp product.
Primers sequence used for detection of N. gonorrhoeae
(Sequence Removed)
'[SEQ ID No.5] (Sequence Removed)
[SEQ ID No.6]


Alternate primers
(Sequence Removed)
[SEQ ID No.7](Sequence Removed)
[SEQ ID No.8]
Amplified region of gene used in diagnosis
(Sequence Removed)
Internal control
Primers sequences used in prototype kit as an internal control.
Primers sequence used for internal control
(Sequence Removed)
[SEQ ID No.9] (Sequence Removed)
[SEQ ID No.10] Alternate sequences
(Sequence Removed)
[SEQ ID No.ll] (Sequence Removed)
[SEQ ID No.12] Amplified region of gene used in diagnosis
(Sequence Removed)

This present invention has been explained in greater detail in the examples:
Example 1:
Prototype kit one for diagnosis of Chlamydia Trachomatis
Kit with internal control.
Protocol 1
Sample collection:
Sample is collected as a swab and is dipped in the vial containing sterile lml Transport medium and stored at 4C for several hours or freezer for a maximum time up to one week.
Preparation of Sample DNA:
1. Take 500µl of collected sample cocktail, in a fresh microcentrifuge tube (1.5ml) and centrifuge at 15,000g, for 30 minute at 4°C.
2. Discard the supernatant carefully without disturbing the pellet.
3. Mixed the appropriate amount of solution (Solution A, 48µl +Solution B, 2µl) before use.
4. Suspend the pellet from step 3 in 50µl of the above solution.
5. Incubated at 37°C for 1hr.
6. Now incubate the sample at 100°C for 10 minute.


7. Spin at 10,000 rpm for 2 minute.
8. Collect the supernatant carefully in separate tube (Designate as Sample DNA).
Setting up the PCR:
Sample DNA 08µl
Reaction mixture I 28µl
Reaction mixture II 16µl
PCR program:
Step Temp Duration
One cycle 1 94°C 5 minutes
35 cycles of 2 95°C 30 seconds
Step 2 to 4, 3 63°C 30 seconds
4 72°C 30 seconds
One cycle 5 72°C 5 minute
6 4°C Tubes can be
taken out after keeping for 10 minute at 4C.
After completing the PCR reaction, add 10µl of the loading dye. Fractionate the product on the agarose gel (1.5%). By loading 25-30 µl
the ample per well as described below.
Preparation of agarose gel: Protocol:


1. To prepare 100 ml of a 1.5% agarose solution, weigh 1.5 g agarose into a glass beaker or flask and add 100 ml IX Running buffer (Dilute from 50x Running buffer before use
2. Microwave or stir on a hot plate until agarose is dissolved and solution is clear.
3. Allow solution to cool to about 55 °C before pouring.
4. Prepare gel tray by sealing ends with tape or other custom made dam.
5. Place comb in gel tray about 1 inch form one end of the tray and position the comb vertically such that the teeth are about 1-2 mm above the surface of the tray.
6. Pour gel solution into tray to a depth of about 5 mm. Allow gel to solidify about 20 minutes at room temperature.
7. To run, gently remove the comb, place tray in electrophoresis chamber, and cover (just until wells are submerged) with IX Running buffer (the same buffer used to prepare the agarose)
8. Load 25-30 µl of PCR product with loading dye per well. Use one lane for loading 5µl of the marker DNA.
9. Electrophoreses at 50-150 volts until orange dye have migrated an appropriate distance (about 5-6 cms.)
10. Excess agraose ca be stored at room temperature and remelted in a microwave and can be used again.
Result analysis:


A. All reaction, give PCR product of 800base pair for internal control.
This proves PCR reaction is working fine and there is no inhibitor
of PCR in the clinical sample.
B. Only Chlamydia trachomatis positive sample, give an extra band of
368bp is evident from the DNA size marker lane.
Alternate protocol for sample preparation from clinical samples.
Protocol 2
Preparation of Sample DNA:
1. Take 500µl of collected sample cocktail, in a fresh microcentrifuge tube (1.5ml) and centrifuge at 15,000g, for 30 minute at 4°C.
2. Discard the supernatant carefully without disturbing the pellet.
3. Suspend the pellet from step 2 in 50µl of TE solution.
4. Now incubate the sample at 100°C for 10 minute
5. Spin at 10, 000 rpm for 2 minute.
6. Collect the supernatant in separate tube (Designated as Sample DNA).
All other steps will be as mentioned in the detailed assay.
COMPONENTS SUPPLEID IN THE KIT ONE:
1. Transport Medium for sample collection
2. Solution A


3. Solution B
4. Reaction mixture I
5. Reaction mixture II
6. Gel loading dye
7. Agarose gel
8. Gel running buffer (50X)
9. DNA marker ladder

10. TE solution
11. Protocol 1
12. Protocol 2
Annexure : 2
Prototype Process two for diagnosis for Chlamydia Trachomatis
Kit II without internal control
Protocol 1
Sample collection:
Sample is collected as a swab and is dipped in the vial containing sterile 1m 1 Transport medium and stored at 4 °C for several hours or in freezer for a (maximum time up to one week).
Preparation of Sample DNA:


1. Take 500µl of collected sample cocktail, in a fresh microcentrifuge tube (1.5ml) and centrifuge a 15, OOOg, for 30 minute at 4°C.
2. Discard the supernatant carefully without disturbing the pellet.
3. Mixed the appropriate amount of solution (Solution A, 48µl +Solution B, 2µl) before use.
4. Suspend the pellet from step 3 in 50µl of the above solution.
5. Incubated at 37°C for 1hr.
6. Now incubate the sample at 100°C for 10 minute
7. Spin at 10,000 rpm for 2 minute.
8. Collect the supernatant carefully in separate tube (Designated as Sample DNA).
Setting up the PCR:
Sample DNA 08µl
Reaction mixture I 26µl
Reaction mixture II 16µl
PCR program:
Step Temp Duration
One cycle 1 94°C 5 minutes
35 cycles of 2 95°C 30 seconds
Step 2 to 4, 3 63°C 30 seconds
4 72°C 30 seconds


One cycle 5 72 °C 5 minute
6 4°C Tubes can be
taken out after keeping for 10 minute at 4°C.
After completing the PCR reaction, add 10µl of the loading dye. Fractionate the product on the agarose gel (1.5%). By loading 25-30 µl the ample per well as described below.
Protocol:
1. To prepare 100 ml of a 1.5% agarose solution, weigh 1.5-g agarose into a glass baker or flask and add 100 ml IX Running buffer. (Dilute from 50xgel running buffer before use)
2. Microwave or stir on a hot plate until agarose is dissolved and solution is clear.
3. Allow solution to cool to about 55°C before pouring.
4. Prepare gel tray bye sealing ends with tape or other custom-made dam.
5. Place cob in gel tray about 1 inch from one end of the tray and position the comb vertically such that he teeth are about 1-2 mm above the surface for the tray
6. Pour gel solution into tray to a depth of about 5 mm. allow gel to solidity about 20 minutes at room temperature.
7. To run, gently remove the comb, place tray in electrophoresis chamber, and cover (just until wells are submerged) with IX Running buffer (the same buffer used to prepare the agarose)


8. Load 25-30 µl of PCR product with loading dye per well. Use one lane for loading 5µl of the marker DNA.
9. Electrophoreses at 50-150 volts until orange dye have migrated an appropriate distance (about 5-6 cms).
10. Excess agarose can be stored at room temperature and remelted in a microwave and can be used again.
Result analysis:
Only Chlamydia trachomatis positive sample give a band of 368 bp as is evident from the DNA size marker lane.
COMPONENTS SUPPLEID IN THE KIT:
1. Transport medium for sample collection
2. Solution A
3. Solution B
4. Reaction mixture I
5. Reaction mixture II
6. Gel loading dye
7. Agarose gel
8. Gel running buffer (50X)
9. DNA marker ladder

10. TE solution
11. Protocol 1
12. Protocol 2

Alternate protocol for sample preparation from clinical samples. Protocol 2 Preparation of Sample DNA:
1. Take 500µl of collected sample cocktail, in a fresh microcentrifuge tube (1.5ml) and centrifuge at 15,000g, for 30 minute at 4°C.
2. Discard the supernatant carefully without disturbing the pellet.
3. Suspend the pellet from step 2 in 50µl of TE solution.
4. Now incubate the sample at 100 C for 10 minute
5. Spin at 10,000 rpm for 2 minutes.
6. Collect the supernatant carefully in separate tube (Designated as Sample DNA.)
All other protocols will be as mentioned in the detailed assay.
Annexure: 3
Prototype Kit III
For diagnosis of Chlamydia Trachmomatis and Nessierria Gonorrhoeae kit III with internal control.
Protocol
Sample collection:


Sample is collected as swab and is dipped in the vial containing sterile lml transport medium and stored at 4C for several hours or in freezer for a (maximum time up to one week).
Preparation of Sample DNA:
1. Take 500µl of collected sample cocktail, in a fresh microcentrifuge tube (1.5ml) and centrifuge at 15,000g for 30 minute at 4°C.
2. Discard the supernatant carefully without disturbing the pellet.
3. Mixed the appropriate amount of solution (solution A, 48µl+ Solution B, 2µl) before use.
4. Suspend the pellet form step 3 in 50µl of the above solution.
5. Incubated at 37°C for lhr.
6. Now incubate the sample at 100°C for 10 minute.
7. Spin at 10,000 rpm for 2 minute.
8. Collect the supernatant carefully in separate tube (Designated as Sample DNA).
Setting up the PCR:
Sample DNA 08µl
Reaction mixture I 26µl
Reaction mixture II 16µl
PCR program:
Step Temp Duration
One cycle 1 94°C 5 minutes


35 cycles of 2 94°C 45 seconds
Step 2 to 4, 3 50°C 45 seconds
4 72°C 45 seconds
One cycle 5 72°C 5 minute
6 4°C Tubes can be
taken out after keeping for 10 minute at 4C.
After completing the PCR reaction, add 10µl of the loading dye. Fractionate the product on the agarose gel (1.5%). By loading 25-30 µl the ample per well as described below.
Protocol:
1. To prepare 100 ml of a 1.5% agarose solution, weigh 1.5-g agarose into a glass beaker or flask and add 100 ml IX Running buffer (Dilute from 50X running buffer by taking 98 ml water and 2 ml of 50X running buffer).
2. Microwave or stir on a hot plate until agarose is dissolved and solution is clear.
3. Allow solution to cool to about 55°C before pouring.
4. Prepare gel tray by sealing ends with tape or other custom made dam.
5. Place comb in gel tray about 1 inch from one end of the tray and position the comb vertically such that the teeth are about 1-2 mm above the surface of the tray.


6. Pour gel solution into tray to a depth of about 5 mm. Allow gel to solidify about 20 minutes at room temperature.
7. To run, gently remove the comb, place tray in electrophoresis chamber, and cove (just until wells are submerged) with IX Running buffer (the same buffer used to prepare the agarose)
8. Load 25-30µl of PCR product with loading dye per well. Use one lane for loading 5µl of the marker DNA.
9. Electrophoreses at 50-150 volts until orange dye have migrated and appropriate distance (about 5-6 cms).
10. Excess agarose can be stored at room temperature and remelted in a microwave and can be sue again.
Result analysis:
A. All reaction, give PCR product of 800 base pair for internal control.
This proves PCR reaction is working fine and there is no inhibitor
of PCR in the clinical sample.
B. Chlamydia trachomatis, Neisseria gonorrhoeae positive sample,
gives two extra band of 368bp for Chlamydia trachomatis and
260bp for Neisseria gonorrhoeae, as is evident from the DNA size a
marker lane.
C. Only Chlamydia trachomatis, positive sample, gives one extra band
of 368 bp. As is evident from the DNA size marker lane.
D. Only Neisseria gonorrhoeae, positive sample, gives one extra band
of 260 bp, as is evident from the DNA size marker lane.


COMPONENTS SUPPLIED IN THE KIT:
1. Transport Medium for sample collection
2. Solution A
3. Solution B
4. Reaction mixture I
5. Reaction mixture II
6. Gel loading dye
7. Agarose gel
8. Gel running buffer (50X)
9. DNA marker ladder
10. Protocol

We Claim
1. A multiplex PCR based process for detecting Chalamydia trachomatis and Neisseria gonorrhoeae, using
i) a reaction mixture having primers, wherein the primers are
Primers sequences of Phospholipase D Endonuclease Superfamily gene , used in diagnosis of Chlamydia trachomatis generates 368-bp product.
Primers sequence used for detection of C. trachomatis (Sequence Removed)
[SEQ ID No.1] (Sequence Removed)
[SEQ ID No.2]
Alternate primers
(Sequence Removed)
[SEQ ID (Sequence Removed)
[SEQ ID No.4]
Primers sequences of Orfl gene used in diagnosis of Neisseria
gonorrhoeae, generates 260-bp product.
Primers sequence used for detection of N. gonorrhoeae
(Sequence Removed)
[SEQ ID No.5]
(Sequence Removed)
[SEQ ID No.6]
Alternate primers
(Sequence Removed)
[SEQ ID No.7] (Sequence Removed)
[SEQ ID No.8]
Internal control
Primers sequences used in prototype kit as an internal control. Alternate sequences
(Sequence Removed)
[SEQ ID No.9] (Sequence Removed)
[SEQ ID No. 10] Preferred sequences
(Sequence Removed)
[SEQ ID No. 11] (Sequence Removed)
[SEQ ID No. 12]
ii) a transport medium of 130 mM Nacl, 2.6mM KCI, lOmM Na2 HP04, 1.7 mM KH2P04, lOmM EDTA, (for sample collection)
iii) solution A of Tris (50 mM), EDTA (ImM) Tritonx 100 (1%) and solution B of proteinase K (200 ug/ml) for sample processing
Wherein the steps comprising
a) collection of sample in one ml of transport medium
b) preparation of sample DNA by taking 500 of collected sample cocktail, centrifuging at 15, 000 g for 30 minute at 4°C
c) discarding the supernatant carefully without disturbing the pellet
d) mixing appropriate amount of solution A 48µl +solution B 2µl
e) suspending the pellet in 50 µl of above solution
f) incubating at 37°C for 1 hr
g) incubating the sample at 100 °C for 10 min
h) Spinning at 10, 000 rpm for 2 min and collecting the supernatant carefully
i) Setting up PCR with 8µl sample DNA, 26µl reaction mixture 1 and 16µl reaction mixture II, wherein the PCR condition is one cycle of 94 °C for 5 minutes followed by 35 cycles of step 2 to step 4 at 95 °C, 63 °C and 72 °C for 30 seconds each and finally one cycle of 72 °C for 5 mins.
2. A multiplex PCR based process for detecting Chlamydia trachomatis and Neisseria gonorrhoeae as claimed in claim 1, wherein Chlamydia trachomatis positive sample gives a band of 368 bp and Neisseria gonorrhoeae sample gives a band of 260 bp.
3. A multiplex PCR based process for detecting Chlamydia trachomatis and Neisseria gonorrhoeae as claimed in claim 1, wherein with the use of internal control, all samples infected or not give an amplicon of 800bp, suggesting no inhibition of PCR assay and infected sample give band of 368 bp alongwith above band of 800 bp.
4. A multiplex PCR based process for detecting Chlamydia trachomatis and Neisseria gonorrhoeae as claimed in claim 1, wherein without internal control, infected sample generates 368 bp product.
5. A multiplex PCR based process kit for detecting Chlamydia trachomatis and Neisseria gonorrhoeae as described and illustrated herein.