AN IMPROVED MULTIPLEX PCR-BASED PROCESS FOR DETECTING
CHALAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAE
FIELD OF INVENTION
The present disclosure relates to an improvement of multiplex PCR-based process for
detecting Chlamydia trachomatis and Neisseria gonorrhoeae. Further, the present
invention relates to a primers and probes useful in detection of various sexually
transmitted pathogens like bacteria, fungi and virus by multiples PCR reaction followed
by visual detection method.
BACKGROUND
Chlamydia trachomatis (CT) and Neissera gonorrhoeae (NG) are causative agent of
common sexuality transmitted diseases, various inflammatory pathologies of the male
and female urogenital systems. If not timely diagnosed, they could lead to infertility,
ectopic pregnancy etc. Gonoccocal and Chlamydia infections have been implicated in
facilitating Human Immunodeficiency Virus acquisition and transmission.Furthermore,
newborns from infected mothers can contract pulmonary and/or ocular infections during
delivery. The pathogenic mechanism of NG involves the attachment of the bacterium to
noncilated epithelial cells via pili. The mechanism also includes the production of
endotoxin and IgA proteases, whereas chlamydiae have a unique biphase life cycle
consisting of a metabolically inactive but infectious extracellular stage and a replicating
but non-infectious intracellular stage. The replicative stage of the life-cycle takes place
within a membrane-bound inclusion the bacteria away from the cytoplasm. of the
infected host cell.
Rapid and specific diagnostic tests are of utmost importance for successful intervention
against CT and NG. Over last 15 years, diagnosis based on selective growth of the
pathogentic bacteria has been the standard, but long time consumption and inhibition of
cell growth of various clinical isolates by various inhibitors make cell culture and
diagnostis on the basis of various biochemical properties less popular. Because bacterial
infection causes antibody production in the host, sera from patients suffering genital tract
infections have also been used to diagnose CT infection. However, assay based on
serological markers are non-quantitative and often difficult to interpret. (Black et al.,
1991; Ngeow, 1996).
Diagnostics on the basis of Polymerase chain reaction (PCR) and DNA hybridization
technique are the current techniques used. PCR is well known process well described in
US patents 4,683, 202, 4683195, 4695188 and current protocols in molecular biology,
(Eds.ausube1, f.M. et a1 (John Wiley & sons; 1995). Further pathogen specific labeled
probes are added to amplified mixtures, and detection occurs if hybridization occurs.
Many patents incorporating these techniques have been issued.
Various kits are commercially available for diagnosis of Neisseria and Chlamydia. These
kits are highly expensive thus are unable to replace the culture method. These kits are
highly expensive thus are unable to replace the culture method. COBAS AMPLICOR NG
(Roche Diagnostic system) has proven to be highly sensitive (92.9 to 100%) and specific
(99 to 100%) fo NG and CT detection. Cost effectiveness, sensitivity of PCR to inhibition
(Roche Diagnostic system) in Indian scenario impedes the success of these kits. The
detection step following PCR in Roche kit is ELISA based and thus is time consuming
and requires expertise.
Abott Laboratories (Abott park, IL) makes Uri Probe, also a test for CT and NG, which
relies on ligase chain reaction (LCR). It uses thermostable ligase enzyme to ligate two
DNA strand, thus generating a detectable ligated DNA fragment only if the template
DNA is present.
None of the above technique uses easy visual assay for the simultaneous detection of
multiple of multiple pathogens. Although Roche cobas amplicor kit is designed to detect
both the pathogens in same sample but have different detection plates. Also the detection
process is ELISA based based and time consuming and requires expertise. Thus such a
test which allows simulataneous amplification and easy detection will be desirable.
Molecular beacons are hair pin shaped oligonucleotides complementary to targeted
nucleic acid sequences having covalently attached 5' flourphore and 3' quencher () used
for the recognition of infectious agents is one alternative to problematic immunological
identification assays and other pre existing methodologies. Thus, are suitable for rapid
diagnostic techniques for the detection of pathogens in clinical samples. Highly specific
probes, able to differentiate between pathogenic and non pathogenic strains are designed
in this study.
OBJECTS OF THE INVENTION
An objective of the present invention is to propose probe based detection of a PCR
product Chlamydia trachomatis or Neisseria gonorrhoeae with an internal control.
Another objective of the present invention is to propose a probe based detection of a PCR
product Chlamydia trachomatis and Neisseria gonorrhoeae with an internal control and
check for the presence of co-infection of two pathogens.
Further object of this invention is to propose a method which can be operated with even
technical expertise.
SUMMARY OF THE INVENTION
The present invention provides methods and reagents for the rapid detection of CT and
NG that are specific, that is, without substantial detection of other species in the
Chlamydia or Neisseria genus or species from other genera. For example the primers will
typically amplify and molecular beacon will hybridize to nucleotide sequence specific to
the nucleotide sequences present in NG but not other specie. Further, since patients,
infected with NG may also be infected with CT; the invention alos provide of
concurrently detecting NG and CT in samples. Thus, minimizing the cost and time
consumption for the diagnosis.
In one aspect, the invention provides an oligonucleotide consisting of a nucleic acid with
a sequence selected from the group consisting of SEQ ID NOS: 1CT to 8CT, 9NG to 12
NG, 131C to 16 IC, and complements thereof, which oligonucleoide has 36 or fewer
nucleotides. Typically, these oligonucleoide are probe nucleic acids comprising of GC
rich arms and fluorescent label at 5' and quencher moiety at 3'.
The invention provides a method for co amplification and detection of Chlamydia
trachomatis and Neisseria gonorrhoeae in a sample, which method includes (a)
contacting nucleic acids from the sample with at least a first pair of primer nucleic acids
that selectively bind to a nucleic acid. The method also includes (b) detecting one or more
arnplicons thereof from the nucleic acid amplification reaction during or after (a), using
molecular beacons thereby detecting the Chlamydia trachomatis and Neisseria
gonorrhoeae in the sample by visualizing the detectable signal produced by the label.
The primer designated SEQ ID No. 1, 2 & 3, 4 amplify (see Indian patent #12/651/819)
and SEQ ID No. 1CT to 4CT detect plde gene of C. trachomatis.The primer designated
SEQ ID No. 5,6 & 7,8 amplify (see Indian patent #I2165 11819) and SEQ ID No.9NG to
12NG detect orfl gene of N. gonorrhoeae. gyraseA is another gene used to amplify
Chlamydia trachomatis (SEQ ID No. GI, G2 & G3, G4) detected by probe SEQ ID No.
5CT to 8CT.
In another creation of invention, primers SEQ ID No. 9, 10 & 11, 12 (see patent #
1216511819) and molecular beacon (13 IC to 16 IC) for an internal control are also
included in may of the methods or kits provided
DETAILED DESCRIPTION OF THE INVENTION
The present invention uses single test to diagnose the multiple pathogens using
specific primers and probes in the same reaction. The test uses multiplex PCR (MPCR
for co amplification (Indian Patent 1216511819) and use of pathogen specific
fluorescently labeled molecular beacon for easy detection of the pathogens.
PCR background
PCR technique is well established and available in texts as in Current protocols in
molecular biology (Martha F. Kramer, Donald M. coen1 unit 15.1), A
polynucleotide, set comprises of a pair of primers and labeled probe. In the context of
the present invention, a polynucleotide is a nucleic acid polymer deoxyribonucleic
acid (DNA) and modified DNA. Modifications include attachment of a label etc.
A primer pair is a pair of oligonucleotide, usually 18-22 nucleotides (nt) in length.
Each primer specifically hybridizes to different strand in an orientation such that the
3'or extendable end of each primer points towards each other.
Preferred primers are without internal homology or primer primer homology.
The term "specifically hybridize" as used herein refers to the ability of primer sets
and molecular beacon to specifically amplify and hybridize with target nucleic acid
strands under and appropriate amplification and hybridization conditions
DNA was isolated from vaginal swabs are obtained from patient samples suspected
of carrying an STD. Primers used in the study are preferably complementary to the
target site. In order to use primers for co amplification, primers are designed such
that these have similar Tm and are able to co amplify under same conditions.
Molecular beacons are used to detect the presence of presence of infection using
visual assay. Briefly, beacon has 15-30 bp long gene specific nucleotide sequence
with no internal secondary structures like internal loops, hairpin loops etc. Two
complementary GC rich arm sequences are added on either side of the probe
sequence. Flurophore and quencher are attached to the 5' and 3' position of the
beacon. Its Tin should be 7C-10 C higher than annealing temperature of primers.
Also probe shouldn't be complementary to primers to cause primer extension. When
no amplicon .is present, beacon would remain in its closed confirmation and would
remain dark. When it binds to specific amplicon, fluorophore and quencher move
apart making beacon to fluoresce under the suitable excitation wavelength (Tyagi S
and Kramer FRl996.www.molecularbeacon.com). Molecular beacon could be added
before, or after PCR) amplification and is allowed to specifically binding to its target
by heating the mix to 95OC followed by slow cooling to 20°C at the ramping rate of
O.l°C/sec.
Detection may include observing a change in fluorescence in a label that is present in
a reporter present in the reaction mixture. Observing a change in fluorescence may
include, observing an increase in fluorescence with the comparison of background
-limit.
In present invention, selection of primers to be used with the molecular beacon probe
is made to meet required criteria. For example, it is important that there are no areas
of complementarities that may cause the molecular beacon to bind to a primer, which
would result in a high background signal.
In present invention, both CT and NG can be amplified and detected simultaneously
in a single reaction mixture using a combination of two primer probe sets .(i.e. one
selected from the CT specific primerlprobe sets and the other, selected from the NG
specific primerlprobe sets). To carry out successful detection of distinct target
sequences, the probes used must be detectably distinct from each other. For example
if two or more molecular beacon probes are present in the same reaction mixture, the
fluorophore moieties of each beacon probe should fluoresce at different wavelengths.
Pre-existing methodology developed by Saluja et.al, Please refer to Indian Patent no
12/631/819 (3437/DEL/2005)
Primers sequences of Phospholipase D Endonuclease Superfamily gene, used in
diagnosis of Chlamydia trachomatis generates 368-bp product whereas an amplicon of
250bp is generated using primer sequences of orfl gene for diagnosis of Neisseria
gonorrhoeae.
Oligos used in invention
Molecular beacon probe complimentary to stretch of Phospholipase D Endonuclease
Superfamily gene used in diagnosis of Chlamydia trachomatis.
Probe sequence
GCATGCG-ACCCCTCCCCTCACACATCTCAAAATA-CGCATGC [SEQ ID NO.
IGCCTATGc-TGGAGAAGGAGTATTTTGAGATGTGTGA-Gcc [sEQ ID NO.
2CT1
GC~TGC-CTTTGCTGATCACTTCTTTTTGTCGTT-GCATGSCE ID No. 3CT]
GcATGc-GTTGTGGTTTGTTTGGTGAGcTGAGTGGA-GcA+Gc~sID NO.
4CT]
Amplified region of gene used in diagnosis
TCTTTTTAAACCTCCGGAACCCACTTCTTCCACAGATTCTTCTAAAGAACC
TCCTAAAGAATCTGCATGGAAAGTAGTCTCTCATTCTCGAGGACGCCGTC
GCGCTCGATCCAACCCCTCCCCTCACACATCTCAAAATACTCCTTCTCCAA
AAGACTCTTCTTTAGTTGCTCGTACGGATAAAGCGGCAACAGATATCTTTA
ATTCGGCTAAACACAAAGCGATTGAAACGACAAAAAGAAGTGATCAGCA
AAGCAGATCCTTACATATACTGCACCTTTTAGCTGAAAATCCGGAACCCA
TTGTGTTCCACTCAGCTCACCAAACAAACCACAACGATCCGCAAAGAATG
CTATGCGATGCCATCC
Primers sequences of gyrase A gene, used in diagnosis of Chlamydia trachomatis
generates 542 bp product.
Preferred primers 5'- CCTGATGCTAGGGACGGATT -3' [SEQ ID No.G1];5'-
CCCTAAATTATGCGGTGGAATA-3'[SEQ ID NO. G2]
Alternate primers 5, - TGATGCTAGGGACGGATTAAAACC -3'[SEQ ID No. G3];
5'- TTCCCCTAAATTATGCGGTGGAA -3' [SEQ ID NO. G4]
Molecular Beacon Probes complimentary to stretch of gyraseA gene used in
diagnosis of Chlamydia trachomatis
GCGAGG-ATAAATGACACTTTCTCCATGAGGGTGATAAT-CCTCGC [SEQ ID
No. 5CTI
GCGAGG-CGCGGTAGGGATGGCAACAAATAT'TC-CCTCGC SEQ ID NO. 6CT]
GCGAGG-ATTCCCCAATTTACTTTGTAATGGCTCCT-CCTC C [SEQ ID NO.
7CT(.
c!
GCGAGG-AACTACGATGAAACTAAA-CCTCG[CS EQ ID N6.8CTI
Amplified region of gene, used in diagnosis
CCTGATGCTAGGGAGGGATTAAAACCTTCTCAGCGACGTATTTTATACGCT
ATGAAACAATTAAATCTGACTCCAGGAGTAAAGCACAGAAAATGCGCAA
AAATTTGCGGTGATACT'TCCGGGAGATTATCACCCTCATGGAGAAAGTGTC
ATTTATCCTACTTTAGTAAGGATGGCACAGGATTGGGCCATGCGATACCCT
TGCCATGCGATATACAGAGGCTCGCCTGACTCACAGCGCTATCTTTTTGTT
AGAGGACCTAGATAAAGATACTGTAGATATGGTCCCTAACTACGATGAAA
CTAAATATGAACCTGTAGTTTTTCCTTCAAAATTCCCCAATTTACTTTGTAA
TGGCTCCTCAGGCATCGCGGTAGGGATGGCAACAAATATTCCACCGCATA
ATTTAGGGGAATTAATTGAGGCTACGCTATTAGTTTTGGCTAA C A G TC A A A
CCTCTATTGAAGATATTTTGGAGGTAATGCCTGGCCC
Molecular Beacon Probe sequences against complimentary sequences of Orfl gene,
used as a target sequence in diagnosis of Neisseria gonorrhoeae
CCAGCG-GATTCCCTTGTCAAGATTTTTCCATGATTT-CGCTGG [SEQ ID No.9NGI
CCAGCG-AATTTGCTGGATGGCTTTTTTCTTGTTGG-CGCTGG SE ID N0.10 NGI
CCAGCG-TTTTTGTTGCTGAGAATGTGAAAGGTTTAT-CGCTG [S Q ID
No.llNGI b %
CCAGCG-TGACTGCCAACAAGAAAAAAGCCATCC-CGCTGG [SEQ ID No.l2NGI
Amplified region of gene used in diagnosis
GCAACTATTCCCGATTGCGACATCATTTTAGGCGGATTCCCTTGTCAAGAT
TTTTCCATGATTTGGAAACAGCCGGGCTTAGAGGGTGAGCGCGGCAATCT
TTATAAAAGCTTTTTACGTTTTGTAAATGCAAAAAAACCGAAAGTTTTGT
TGCrGAGAATGTGAAAGGTTTATTGACTGCCAACAAGAAAAAAGCCATCC
AGCAATTATTACCGACTTTGAAAATTGCGGTTATTACGTTCAGGCGAAG
CTGTATAAC
Internal control
Probes sequences used in prototype kit as a internal control.
GCGAGG-GTCTTGTCGTGCTCGTTCTTCT-CCTCGCS EQ ID NO.1 31C GCGAGG-CACTTGGGCTGCTGGTAGGTTTTGGG-C TCGCE S EQ ID I.10 .141CI
GCGAGG-TTTTACCCCCAGGAAGGACTTGAACTGT-CCTC C rSEO ID NO. L . 15IC(
GCGAGG-TTTTCATACACCTCCTGGCTCTTCAGCAC-CCTCGC ISEQ ID NO.
16ICI
Amplified region of gene used in diagnosis
CGTACCAGAAGGAGCAGCTGAACACGAGGGGCCGGCCATTCCGAGGCAT
GTCTGAAGAGGAGGTGTTCACCGAGGTGGCCAACCTCTTCCGGGGCCAGG
AGGACCTGCTCTCAGAGTTTGGACAGTTCCTGCCCGAAGCCAAGCCGGTCT
CTGTTCACAGGAAACGGGCCGTGCGAGATGCACAGCGTGCAGAAGAACG
AGCACGACAAGACCCCGGAGCACAGCAGGAAGCGCTCCCGGCCCI'CGCT
CCTCCGCCCCGTGTCTGCACCCGCCAAGAAAAAAATGAAACTTCGTGGTA
TTTTCTTTCTTTGACAAGGTCCGCCGGGTGCTGAAGAGCCAGGAGGTGTAT
GAAAACTTCCTCCGCTGCATCGCACTC'ITCAACCAGGAGCTGGTGTCTGGC
TCTGAGCTCCTGCAGCTCGTCAGCCCATTTCTGGGGAAAT'I-TCCAGAACTC
TTTGCACAGTTCAAGTCCTTCCTGGGGGTAAAAGAGCTGTCCTTCGCGCCA
CCCATGAGCGACAGATCCGGGGACGGGATAAGCCGGGAAATTGATTATG
CATCCTGCAAGCGCATAGGATCCAGCTACCGGGCACTCCCCAAAACCTAC
CAGCAGCCCAAGTGCAGTGGGAGGACAGCCATCTGCAAGGAGCTTGACC
ATTGGACACTTCTCCAGGGTTCGTGGACAGACGATTACTGCATGTCCAA
TTCAAGAATACCTGCTGGATTCCAGGATATAGTGCAGGGGTAGTGAACGA
CACCTGGGTCTCCTTCCCTTCCTGGTCTGAGGACTCCACGTTCGTCAGCTC
CAAGAAGACACCGTACGAGGAGCAGCTTCACCGCTGTGAGGACGAGCGC
TTCGAGTTAGACGTTGTCCTGGAGACG
In principal any pair of oligonucleotide which binds to the two strand of duplex DNA
in sufficiently close proximity would be used as primers for PCR but in practice no
guarantee of success in selecting suitable primer set for PCR. However it is known
that many theoretically suitable primer sets simply do not work in practice for coamplification.
To enhance the specificity, hair pin shaped probes having a
fluorophore and quencher covalently attached to respective arms is used. It fluoresces
when binds to specific amplicon, separating quencher and flourophore and thus
restoring fluorescence. This step also adds another level of .specificity as if the
amplicon is non-specific, the beacon will fail to hybridize to amplicon internally and
hence no fluorescence will be observed.
The primer sets identified by our laboratory have been established that they are
specific and complimentary to the target nucleic acid to form a desired hybridization
product and then be extendable by DNA polymerase in practical under routine use. The
fluorescently labeled hairpin shaped probe further enhances specificity and
sensitivity.
However, we claim that we are using exactly complimentary primer sets and labeled
probes to the target site of genomic DNA sequences of Neisseria gonorrhoeae and
Chlamydia trachomatis For designing a diagnostic method using crude samples while
cryptic plasmid is being used in most of the existing methods.
So the novelty of method claims identification of unique, specific, sensitive yet cost
effective PCR based detection of Gonococcal and Chlamydia1 infection. Hence claims are
inventive and novel.
General information:
1. INFORMATION FOR [SEQ ID No. 1 CT]
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 1 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D)TOPOLOGY: Hairpin shaped Stem loop
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 1 CT]
GCATGCG-ACCCCTCCCCTCACACATCTCAAAATA-CGCATGC
2. INFORMATION FOR [SEQ IDNo.2CTI
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 1 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 2CT]
GCATGC-TTGGAGAAGGAGTATTTTGAGATGTGTGA-GCATGC
3. INFORMATION FOR [SEQ ID No.3CTI
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 3CT]
GCATGC-TGGAGAAGGAGTATTTTGAGATGTGTGA-GCATGC
4. INFORMATION FOR [SEQ ID No. 4CT]
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 1 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
@) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 4CT]
GCATGC-GTTGTGGTTTGTTTGGTGAGCTGAGTGGA-GCATGC
5. INFORMATION FOR [SEQ ID No. 5CTJ SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D)TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 5CT]
GCGAGG-ATAAATGACACTTTCTCCATGAGGGTGATAAT-CCTCGC
6. INFORMATION FOR [SEQ ID No. 6CT] SEQUENCE
CHARACTERISTICS:
(A)LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: stem loop
(D)TOPOLOGY: Hairpin shaped
(E)SEQUENCE DESCRIPTION: [SEQ ID No. 6CT]
GCGAGG-CGCGGTAGGGATGGCAACAAATATTC-CCTCGC
7. INFORMATION FOR [SEQ ID No.7CTI
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 1 base pairs
(El) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 7CT]
GCGAGG-ATTCCCCAATTTACTTTGTAATGGCTCCT-CCTCGC
8. INFORMATION FOR [SEQ ID No. 8CT]
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D)TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No.8CTI
GCGAGG-AACTACGATGAAACTAAA-CCTCGC
9. INFORMATION FOR [SEQ ID No.9NGI
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No.9NGI
CCAGCG-GATTCCCTTGTCAAGATTTTTCCATGATTT-CGCTGG
10. INFORMATION FOR [SEQ ID No.lONG]
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 1 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. IONG]
CCAGCG-AATTTGCTGGATGGCTTTTTTCTTGTTGG-CGCTGG
1 1 .INFORMATION FOR [SEQ ID No. 1 ING]
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs 7
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped Stem loop
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 1 ING]
CCAGCG-TTTTTGTTGCTGAGAATGTGAAAGGTTTAT-CGCTGG
12. INFORMATION FOR [SEQ ID No. 12NGl
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Stem loop.
(D) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 12NGl
CCAGCG-TGACTGCCAACAAGAAAAAAGCCATCC-CGCTGG
13. INFORMATION FOR [SEQ ID No. 13ICl
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: Labelled nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped
( ~SEjQ UENCE DESCRIP[STEQI OID NNO:. 131~1
GCGAGG-GTCTTGTCGTGCTCGTTCTTCT-CCTCGC
14.INFORMATION FOR [SEQ ID No. 14ICl
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: Labelled nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 14ICl
GCGAGG-CACTTGGGCTGCTGGTAGGTTTTGGG-CCTCGC
15.INFORMATION FOR [SEQ ID No. 15ICI
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: Labelled nucleic acid
(C)STRANDEDNESS: Stem loop .(D)
TOPOLOGY: Hairpin shaped
(E) SEQUENCE DESCRIPTION: [SEQ ID No. 15ICl GCGAGG-
'ITTTACCCCCAGGAAGGACTTGAACTGT-CCTCGC
16.INFORMATION FOR [SEQ ID No. 16ICl
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 1 base pairs
(B) TYPE: Labelled nucleic acid
(C) STRANDEDNESS: Stem loop
(D) TOPOLOGY: Haimin shaued
(E) SEQUENCE DES~RIPTION[:S EQ ID No. 161Cl
GCGAGG-TTTTCATACACCTCCTGGCTCTTCAGCAC-CCTCGC

We Claim:
1. An improved multiplex PCR-based process for detecting Chlamydia trachomatis and
neissera gonorrhoeae, wherein the improvement comprises a method of:
a) visualization assay for simultaneous amplification and detection without an
internal control; and
b) visualization assay for simultaneous amplification and detection with an internal
control.
2. The said improvement according to Claim 1 enhances specificity and sensitivity of the
process.
Dated this 19'~d ay of November, 2013