Method for detecting telomerase via washing-free anchored-extension and telomeric-binding amplification, and kit

Abstract

Provided is a new method for telomerase amplificationwashing-free anchored-extension and telomeric-binding amplification (WATA). The method comprises: utilizing an anchored telomerase primer to conduct telomere TTAGGGG sequence (G sequence) extension; hybridizing with a template probe having a universal PCR primer sequence and six units of telomere CCCTAA sequence (C sequence) on the extended G sequence; removing the uncombined template probe via an enzyme digestion reaction; and the template probe combined with the G sequence conducts PCR reaction, the amplification product being a peculiar DNA fragment of a fixed length.

Claims

1. A method for detecting telomerase activity by washing-free, anchored-extension and telomeric-binding amplification comprising: adding a cell lysate supernatant of a sample to be tested to a PCR tube with a telomerase primer fixed thereto; placing the PCR tube on ice to bind telomerase to the telomerase primer; aspirating off the cell lysate supernatant from the PCR tube, adding to the PCR tube a telomerase reaction system and then incubating the PCR tube at 30-37 C. to perform a telomeric G sequence extension reaction; incubating the PCR tube at 55-65 C. to perform a hybridization reaction; aspirating off supernatant from the PCR tube, adding to the PCR tube a PCR reaction solution containing a restriction endonuclease and PCR primers, and incubating the PCR tube at 37 C. for 10 min; and carrying out a PCR reaction to be analyzed for detection of the telomerase activity, wherein the telomerase primer has a sequence of: TABLE-US-00029 (SEQIDNO:1) 5-TCCGTCGAGCAGAGTTAGGGTTAG-3, and the PCR primers are: TABLE-US-00030 (SEQIDNO:4) 5-CCGTCACCCTGGATGCTGTAGG-3, and (SEQIDNO:5) 5-AAGAGCCGCGAGCGATCCTT-3.

2. The method of claim 1, wherein the method further comprises: (1) synthesizing the telomerase primer and fixing the telomerase primer to the PCR tube; (2) synthesizing a template probe and an inhibitory probe, respectively; (3) complexing the template probe and the inhibitory probe to form a double-stranded DNA designated as dsTU; (4) adding a lysis buffer to the sample to be tested to form a mixture, and repeatedly pipetting the mixture, and then transferring the mixture to a centrifuge tube; (5) placing the centrifuge tube on ice for 10 min, and centrifuging the centrifuge tube at 4 C. to obtain supernatant to be used as a lysate supernatant; (6) adding the lysate supernatant to the PCR tube, placing the PCR tube on ice for 30 min; (7) aspirating off liquid in the PCR tube, thereafter adding to the PCR tube a telomerase reaction solution, and then incubating the PCR tube at 30-37 C. for 30-60 min and at 55-65 C. for 15-30 min; (8) aspirating off liquid in the anchor PCR tube, thereafter adding to the PCR tube a restriction endonuclease/PCR reaction solution, and then performing a PCR reaction, wherein the template probe has a sequence of: TABLE-US-00031 the inhibitory probe sequence has a sequence of: TABLE-US-00032 (SEQIDNO:3) 5-TAGGGTTAGGGATCCTACA-3.

3. The method of claim 2, wherein the telomerase reaction solution in Step (7) has a composition of: 0.06 mmol/L of dNTP, 1 nmol/L of dsTU, 1.5 mmol/L of MgCl.sub.2, 63 mmol/L of KCl, 1 mmol/L of EGTA-Na, 0.1 mg/ml of BSA, 0.05% of Tween 20, and 20 mmol/L of Tris-HCl with pH 8.0 as a solvent.

4. The method of claim 2, wherein the restriction endonuclease/PCR reaction solution in Step (8) has a composition of: 0.2 mmol/L of dNTP, 1 U/50 l of Taq enzyme, 0.2 mol/L of PCR primer, 0.4SYBR Green I, 2 U/50 l of BamHI, 50 mmol/L of KCl, 1.5 mmol/L of MgCl.sub.2, 0.05% of Tween 20, and 10 mmol/L of Tris-HCl with pH 9.0 as a solvent.

5. A kit for detecting telomerase activity by washing-free, anchored-extension and telomeric-binding amplification, comprising an anchor PCR tube fixed with a telomerase primer, a template probe, an inhibitory probe, PCR primers, a telomerase reaction buffer, a restriction endonuclease/PCR reaction buffer, a Taq enzyme and a BamHI enzyme, wherein the telomerase primer has a sequence of: TABLE-US-00033 (SEQIDNO:1) 5-TCCGTCGAGCAGAGTTAGGGTTAG-3; the template probe has a sequence of: TABLE-US-00034 the inhibitory probe has a sequence of: TABLE-US-00035 (SEQIDNO:3) 5-TAGGGTTAGGGATCCTACA-3, the PCR primers are: TABLE-US-00036 (SEQIDNO:4) 5-CCGTCACCCTGGATGCTGTAGG-3, and (SEQIDNO:5) 5-AAGAGCCGCGAGCGATCCTT-3.

6. The kit of claim 5, wherein the kit further comprises a cell lysis buffer with a composition of: 1 mmol/L of MgCl.sub.2, 1 mmol/L of EGTA-Na, 1% of NP-40, 0.25 mmol/L of sodium deoxycholate, 150 mmol/L of NaCl, 10% of glycerol, 5 mmol/L of 2-mercaptoethanol, 0.1 mmol/L of AEBSF, and 10 mmol/L of Tris-HCl with pH 7.5 as a solvent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic process flow chart of a method according to the present invention, which includes:

(2) Step S0: a telomerase primer (TS) is fixed;

(3) Step S1: the cells are lysed to obtain a lysate supernatant containing telomerase molecules;

(4) Step S2: the lysate supernatant is placed on ice to adsorb and bind the telomerase molecules to the TS;

(5) Step S3: after aspiration off of liquid, the telomerase reaction system is added, and subjected to the telomere extension reaction and then to the hybridization reaction, such that a template probe is bound to the telomere extension product;

(6) Step S4: after aspiration off of liquid, a RE/PCR system is added, and incubated at 37 C. before the PCR reaction procedure, so as to cleave the remaining template probe, where the template probe bound to the telomere extension product is kept intact because the enzymatically cleavable site is single-stranded and cannot be cleaved; and

(7) Step S5: the PCR procedure is carried out and the template probe is amplified, indicating the telomerase activity.

(8) TS, telomerase primer; TPB, template probe; CSC, inhibitory probe; RS, restriction site.

DETAILED DESCRIPTION OF THE INVENTION

(9) The present invention is described in further detail with reference to specific examples. However, the protection scope of the present invention is not limited thereto.

Example 1: Removal of the Interference with PCR from a Small Amount of dsTU by a WATA System Integrated with the BamHI Enzyme

(10) (1) Synthesis of the primer and probe sequences as follows:

(11) The sequence of the telomerase primer S is:

(12) TABLE-US-00011 5-TCCGTCGAGCAGAGTTAGGGTTAG-3.

(13) The sequence of the telomerase primer S is:

(14) TABLE-US-00012 (SEQIDNO:1) 5-TCCGTCGAGCAGAG -3.

(15) The sequence of the template probe T is:

(16) TABLE-US-00013

(17) The sequence of the inhibitory probe U is:

(18) TABLE-US-00014 (SEQIDNO:3) 5-TAGGGTTAG TACA-3.

(19) The sequences of the PCR primers are:

(20) TABLE-US-00015 (SEQIDNO:4) 5-CCGTCACCCTGGATGCTGTAGG-3, and (SEQIDNO:5) 5-AAGAGCCGCGAGCGATCCTT-3.

(21) (2) Preparation of anchor PCR tubes fixed with the telomerase primer S:

(22) 50 l of TBST buffer containing 5 pmol biotinylated telomerase primer S was charged to a 0.2 ml Thin-wall PCR tube coated with streptavidin and stood at room temperature for 1 hr. The liquid in the tube was aspirated off, and 100 l of the TBST buffer was added, pipetted, and aspirated off. The tube was repeatedly washed 3 times, and then aspirated off. 100 l of TE buffer was added and the liquid in the tube is then aspirated off. The tube was sealed and stored at 20 C. for later use.

(23) (3) Compositions of the lysis buffer and the reaction solution

(24) The lysis buffer had a composition of: 1 mmol/L MgCl.sub.2, 1 mmol/L EGTA-Na, 1% (vol/vol) NP-40, 0.25 mmol/L sodium deoxycholate, 150 mmol/L NaCl, 10% (vol/vol) glycerol, 5 mmol/L 2-mercaptoethanol, 0.1 mmol/L AEBSF, and 10 mmol/LTris-HCl (pH7.5) as the solvent.

(25) The telomerase reaction solution (the reaction solution T) had a composition of: 0.06 mmol/L dNTP, 1 nmol/L dsTU, 1.5 mmol/L MgCl.sub.2, 63 mmol/L KCl, 1 mmol/L EGTA-Na, 0.1 mg/ml BSA, 0.05% Tween 20, and 20 mmol/L Tris-HCl (pH 8.0) as the solvent.

(26) The RE/PCR reaction solution had a composition of: 0.2 mmol/L dNTP, 1 U/50 l of Taq enzyme, 0.2 mol/L PCR primers, 0.4SYBR Green I, 2 U/50 l BamHI, 50 mmol/L KCl, 1.5 mmol/L MgCl.sub.2, 0.05% Tween 20, and 10 mmol/L Tris-HCl (pH 9.0) as the solvent.

(27) The conventional BamHI free PCR reaction solution had a composition of: 0.2 mmol/L dNTP, 1 U/50 l Taq enzyme, 0.2 pmol/L PCR primers, 0.4SYBR Green I, 50 mmol/L KCl, 1.5 mmol/L MgCl.sub.2, 0.05% Tween 20, and 10 mmol/L Tris-HCl (pH 9.0) as the solvent.

(28) Human lung cancer cell line A549 (commercially available from ATCC, US) was cultured at a density of 10000 cells/well in 8 wells in a 24-well plate. After overnight culture, the media was aspirated off. 200 l of the lysis buffer T was added per well, repeatedly pipetted, transferred to 8 centrifuge tubes of 1.5 ml respectively, placed on ice for 10 min, and centrifuged at 4 C. and 15000 rpm for 10 min. The supernatant was removed and used as the lysate supernatant.

(29) 50 l of the lysate supernatant was added to 8 reaction tubes of 0.2 ml fixed with the telomerase primer respectively, 4 of which were then added with 1 nmol/L dsTU respectively, and the other 4 of which were added with ddH.sub.2O control in a volume equal to that of dsTU. After incubation at 30 C. for 30 min and then at 60 C. for 30 min, the liquid was aspirate off. The BamHI free conventional PCR reaction solution was added to 4 tubes (2 of which contained dsTU, and the other 2 of which contained the ddH.sub.2O control, the same below), and the RE/PCR reaction solution containing BamHI was added to the other 4 tubes. The amplification was carried out on a fluorescent quantitative PCR instrument at 37 C. for 10 min, and 94 C. for 3 min, followed by 40 cycles of 94 C. for 5 sec and 63 C. for 30 sec, and the amplification product was analyzed by SYBR green I fluorescent quantitative assay. The result is shown below.

(30) TABLE-US-00016 Reaction system Ct-dsTU Ct-ddH.sub.2O Conventional PCR 20.72 29.38 Conventional PCR 20.53 29.49 RE/PCR 29.21 29.55 RE/PCR 29.05 29.42

(31) It can be seen that the Ct value (resulting from the primer dimer) of the ddH.sub.2O control in the 2 systems is greater than 29. As to the conventional PCR, the Ct value of the dsTU tube is less than 21, which is far below the value of the control, suggesting that after the lysate supernatant containing 1 nmol/L dsTU in the reaction tube is aspirated off, the remaining trace amount of dsTU is considerably amplified. The Ct value of the RE/PCR dsTU tube is greater than 29, which is comparable with the value of the control, suggesting that the background arising from the amplification of the remaining small amount of dsTU can be effectively controlled by the RE/PCR system through cleavage.

Example 2: Removal of Interference Resulting from Contamination with a Small Amount of PCR Product by a WATA System Integrated with the BamHI Enzyme

(32) The primer and probe sequences, the reaction tube, the lysis buffer T, the reaction solution T, the conventional PCR reaction solution and the RE/PCR reaction solution were the same as those in Example 1.

(33) The A549 cells and the preparation of the lysate supernatant were the same as those in Example 1. 50 l of the lysate supernatant was added to 8 reaction tubes of 0.2 ml fixed with the telomerase primer respectively, 4 of which were then added with the amplification product at a dilution of 10.sup.7 respectively, and the other 4 of which were added with equal volume of ddH.sub.2O control. After incubation at 30 C. for 30 min and then at 60 C. for 30 min, the liquid was aspirate off. The BamHI free conventional PCR reaction system was added to 4 tubes (2 of which contained the amplification product, and the other 2 of which contained the ddH.sub.2O control, the same below), and the RE/PCR reaction system containing BamHI was added to the other 4 tubes. The amplification was carried out on a fluorescent quantitative PCR instrument at 37 C. for 10 min and 94 C. for 3 min, followed by 40 cycles of 94 C. for 5 sec and 63 C. for 30 sec, and the amplification product was analyzed by SYBR green I fluorescent quantitative assay. The result is shown below.

(34) TABLE-US-00017 Reaction system Ct-Product Ct-ddH.sub.2O Conventional PCR 15.39 29.54 Conventional PCR 15.11 29.81 RE/PCR 29.15 29.67 RE/PCR 29.03 29.50

(35) It can be seen that the Ct value (resulting from the primer dimer) of the ddH.sub.2O control in the 2 systems is greater than 29. As to the conventional PCR, the Ct value of the amplification product tube is less than 16, which is far below the value of the control, suggesting that the amplification product at a dilution of 10.sup.7 can cause contamination to the conventional PCR reaction system, which results in serious false positive results. The Ct value of the RE/PCR amplification product tube is greater than 29, which is comparable with the value of the control, suggesting that the false positive results arising from the contamination with the small amount of amplification product can be effectively controlled by the RE/PCR reaction system through cleavage.

Example 3: Detection of the Telomerase Activity in Human Lung Cancer A549 Cells by the WATA Method

(36) The primer and probe sequences, the reaction tube, the lysis buffer T, the reaction solution T, and the RE/PCR reaction solution were the same as those in Example 1.

(37) The detection method was carried out as follows:

(38) (1) The A549 cells were cultured in a 24-well plate (at a density of about 10-10000 cells/well), and then the media was aspirated off. 200 l of the lysis buffer T was added per well, repeatedly pipetted, transferred to a 1.5 ml centrifuge tube, placed on ice for 20 min, and centrifuged at 4 C. and 15000 rpm for 20 min. The supernatant was removed and used as the lysate supernatant.

(39) (2) 50 l of the lysate supernatant was added to the anchor PCR tube, placed on ice for 30 min, and aspirated off, and then 50 l of the reaction solution T containing 1 nmol/L dsTU was added, and incubated at 30 C. for 30 min and then at 60 C. for 30 min.

(40) (3) After aspiration off of the liquid, 50 l of the RE/PCR reaction system (containing the PCR buffer, 0.2 mol/L PCR primers, 0.2 mmol/L dNTP, 1 U Taq enzyme, and 2 U BamHI) was added. The amplification was carried out on a PCR instrument at 37 C. for 10 min and 94 C. for 3 min, followed by 40 cycles of 94 C. for 5 sec and 63 C. for 30 sec, and the amplification product was analyzed by SYBR green I fluorescent quantitative assay.

(41) (4) The lysis buffer and the thermally deactivated (at 80 C. for 10 min) lysate supernatant of 1000 cells were used as the negative control, and in Steps 2)-3) in place of the lysate supernatant.

(42) (5) When the Ct value was 28, a positive result was determined, and when the Ct value was >28, a negative result was determined.

(43) The result is shown below.

(44) TABLE-US-00018 Sample Ct Result Lysis buffer 29.55 Negative 10 cells 27.76 Positive 100 cells 24.19 Positive 1000 cells 20.83 Positive 10000 cells 17.21 Positive Thermally deactivated 29.38 Negative lysate supernatant

(45) It can be seen that the telomerase activity in 10-10000 A549 cells can be detected by the WATA method.

Example 4: Stability of Reaction Tubes Fixed with the Telomerase Primer Prepared in Large Quantity

(46) The primer and probe sequences, the reaction tube, the lysis buffer T, the reaction solution T, and the RE/PCR reaction solution were the same as those in Example 1.

(47) (1) 24 reaction tubes were divided into 6 groups, each having 4 tubes. After sealing, the tubes were stored at 37 C. for 1, 2, 3, 4, 5, and 6 days, and recorded as Groups 1, 2, 3, 4, 5, and 6.

(48) (2) The lysate supernatant (21 ml) of the A549 cells was prepared following the method in Example 1, where 1 ml was placed on ice and ready for use as a positive control; and the other 1 ml was thermally deactivated (at 80 C. for 10 min) and used as a negative control.

(49) (3) The operations were as described in Steps 2) and 3) in Example 3, each group of the reaction tubes included 2 positive controls, and 2 negative controls.

(50) (4) When the Ct value was 28, a positive result was determined, and when the Ct value was >28, a negative result was determined.

(51) The result is shown below.

(52) TABLE-US-00019 Ct-Positive Ct-Positive Ct-Negative Ct-Negative Group control 1 control 2 control 1 control 2 1 17.28 17.43 29.14 29.23 2 17.52 17.39 29.26 29.38 3 17.33 17.45 29.41 29.29 4 17.75 17.54 29.32 29.46 5 24.79 21.93 29.66 29.31 6 27.50 28.25 29.81 29.65

(53) It can be seen that after storage at 37 C. for 1-4 days, the reaction tubes fixed with the telomerase primer prepared in large quantity substantially have no influence on the detection results of the telomerase activity, but have a serious influence on the detection results after storage at 37 C. for 5 or more days.

Example 5: Detection of the Telomerase Activity in Human Squamous Cell Carcinoma Cell Line A431 by the WATA Method

(54) The primer and probe sequences, the reaction tube, the lysis buffer T, the reaction solution T, and the RE/PCR reaction solution were the same as those in Example 1.

(55) The detection method was as follows.

(56) The A431 cells were cultured in a 24-well plate (at a density of about 10-10000 cells/well), and then the media was aspirated off. 200 l of the lysis buffer T was added per well, repeatedly pipetted, transferred to a 1.5 ml centrifuge tube, placed on ice for 20 min, and centrifuged at 4 C. and 15000 rpm for 20 min. The supernatant was removed and used as the lysate supernatant. The following operations were the same as those in Example 3. When the Ct value was 28, a positive result was determined, and when the Ct value was >28, a negative result was determined.

(57) The results are shown below.

(58) TABLE-US-00020 Sample Ct Result Lysis buffer 29.82 Negative 10 cells 27.66 Positive 100 cells 24.91 Positive 1000 cells 21.26 Positive 10000 cells 17.68 Positive Thermally sterilized 29.53 Negative lysate supernatant

(59) It can be seen that the telomerase activity in 10-10000 A431 cells can be detected by the WATA method.

Example 6: WATA Detection Kit

(60) A kit of 8 Tests had a composition as shown below.

(61) TABLE-US-00021 Packing Component Function specification 1) Concentrated Pretreatment of the sample, 1 1.7 ml pretreatment and collection, enrichment, and buffer washing of the cells 50-fold diluted with ddH.sub.2O before use 2) Lysis buffer T Lysis of cells 1 1.7 ml Direct use 3) Reaction Telomeric G sequence extension 1 0.45 ml solution T reaction and hybridization and binding of the template probe Direct use 4) RE/PCR Enzymatic cleavage and PCR 1 0.45 ml reaction solution proliferation Direct use 5) Negative control Thermally deactivated lysate 1 0.12 ml supernatant of 1000 A549 cells Direct use 6) Positive control Lysate supernatant of 1000 1 0.12 ml freshly frozen A549 cells Direct use 7) Reaction tube Fixed with telomerase primer 1 8 Direct use 8) Membrane seal Sealing the reaction tube 3 Direct use 9) Instruction Operation instructions and 1 notices

(62) The pretreatment buffer had a composition of: 50PBS+50 g/L DTT, and had a composition of 1PBS+1 g/L DTT after 50-fold dilution. The composition of 1PBS was NaCl 137 mM, KCl 2.7 mM, Na.sub.2HPO.sub.410 mM, KH.sub.2PO.sub.4 1.8 mM, and distilled water as the solvent.

(63) The primer sequence, the lysis buffer T, the reaction solution T, and the RE/PCR reaction solution were the same as those in Example 1.

(64) The operation steps were carried out as follows:

(65) (1) The kit was removed from a frozen storage environment, thawed at room temperature, and temporarily stored at 4 C.

(66) (2) The concentrated pretreatment buffer was 50-fold diluted with 83 ml ddH.sub.2O, to give a pretreatment buffer that was temporarily stored at 4 C.

(67) (3) A sample was collected, which might be cultured cells and tissues, or sputum, whole blood (supplemented with an anticoagulant), and urine.

(68) The amount of the sample was recommended to be 10-10.sup.6 cultured cells, about 0.1 cm.sup.3 tissue mass, about 2 ml sputum, about 0.5 ml whole blood, and about 10 ml urine.

(69) The sample was pretreated as follows. The cells cultured in suspension were collected by centrifugation, and then re-suspended in 10 ml of the pretreatment buffer. The tissue mass was soaked in 10 ml of the pretreatment buffer, and chopped. The sputum was agitated with 10 ml of the pretreatment buffer at 37 C. for about 10 min until the sputum was completely dissolved. 10 ml of the pretreatment buffer was directly added to the whole blood. After centrifugation of the urine, the pellet was re-suspended in 10 ml of the pretreatment buffer. The materials obtained above were all further centrifuged, the supernatant was discarded, and the pellet was carried on next step.

(70) (4) The lysis buffer T was added to the pellet in an amount of 200 l/sample, repeatedly pipetted, transferred to a 1.5 ml centrifuge tube, placed on ice for 20 min, and centrifuged at 4 C. and 15000 rpm for 20 min. The supernatant was removed and used as the lysate supernatant.

(71) (5) 50 l of the lysate supernatant was added to the anchor PCR tube, placed on ice for 30 min, and aspirated off. 50 l of the reaction solution T containing 1 nmol/L dsTU was added and incubated at 30 C. for 30 min and then at 60 C. for 30 min

(72) (6) The liquid was aspirated off, and then 50 l of the RE/PCR reaction system (containing the PCR buffer, 0.2 mol/L PCR primers, 0.2 mmol/L dNTP, 1 U Taq enzyme, and 2 U BamHI) was added. The amplification was carried out on a PCR instrument at 37 C. for 10 min and then 94 C. for 3 min, followed by 35 cycles of 94 C. for 5 sec and 63 C. for 30 sec, and the amplification product was analyzed by SYBR green I fluorescent quantitative assay.

(73) (7) A negative control and a positive control were set in place of the lysate supernatant, and used in Steps 5)-6).

(74) (8) When the Ct value was 28, a positive result was determined, and when the Ct value was >28, a negative result was determined.

Example 6: Detection of the Telomerase Activity in Human Embryonic Kidney Cell Line 293T with the WATA Detection Kit for Telomerase Activity

(75) The 293T cells (commercially available from ATCC, US) were cultured in a 24-well plate (at a density of about 1-10.sup.6 cells/well). The kit and operation steps were as described in Example 5. The lysate supernatant of 1000 cells was thermally deactivated as described in Example 3.

(76) The result is shown below.

(77) TABLE-US-00022 Sample/Control Ct Results 1 cells 29.10 Negative 10 cells 27.03 Positive 100 cells 23.46 Positive .sup.10.sup.3 cells 20.18 Positive .sup.10.sup.4 cells 16.79 Positive .sup.10.sup.5 cells 14.35 Positive .sup.10.sup.6 cells 14.82 Positive Negative control 29.67 Negative Positive control 20.44 Positive

(78) It can be seen that the telomerase activity in 10-10.sup.6 293 T cells can be detected by the WATA kit. However, when the cell number in the sample reaches to or exceed 10.sup.5, the Ct value does not decline obviously, suggesting that the amplification limiting factor varies from the telomeric G sequence extension to the template probe, that is, the template probe is completely adsorbed and bound. Therefore, although a higher telomerase activity leads to more telomeric G sequence extension, the number of the PCR template is not increased.

Example 7: Detection of the Telomerase Activity in Human Cervical Cancer Cell Line HeLa with the WATA Detection Kit for Telomerase Activity

(79) The Hela cells (commercially available from ATCC, US) were cultured in a 24-well plate (at a density of about 10-10.sup.5 cells/well). The kit and operation steps were as described in Example 5. The lysate supernatant of 1000 cells was thermally deactivated as described in Example 3.

(80) The result is shown below.

(81) TABLE-US-00023 Sample/Control Ct Results 10 cells 26.88 Positive 100 cells 23.37 Positive .sup.10.sup.3 cells 20.09 Positive .sup.10.sup.4 cells 16.55 Positive .sup.10.sup.5 cells 14.44 Positive Negative control 29.59 Negative Positive control 20.32 Positive Thermally deactivated 29.66 Negative lysate supernatant

(82) It can be seen that the telomerase activity in 10-10.sup.6 Hela cells can be detected by the WATA kit, and in the range, the higher the cell number is, the lower the Ct value is, suggesting that the WATA detection for telomerase activity is of great value in quantitative detection.

Example 8: Detection of the Telomerase Activity in Human Breast Cancer Cell Line MCF-7 with the WATA Detection Kit for Telomerase Activity

(83) The MCF-7 cells (commercially available from ATCC, US) were cultured in a 24-well plate (at a density of about 10-10.sup.5 cells/well). The kit and operation steps were as described in Example 5. The lysate supernatant of 1000 cells was thermally deactivated as described in Example 3.

(84) The result is shown below.

(85) TABLE-US-00024 Sample/Control Ct Results 10 cells 27.92 Positive 100 cells 24.43 Positive .sup.10.sup.3 cells 21.27 Positive .sup.10.sup.4 cells 17.88 Positive .sup.10.sup.5 cells 15.29 Positive Negative control 29.33 Negative Positive control 20.47 Positive Thermally deactivated 29.73 Negative lysate supernatant

(86) It can be seen that the telomerase activity in 10-10.sup.5 MCF-7 cells can be detected by the WATA kit, and in the range, the higher the cell number is, the lower the Ct value is, suggesting that the WATA detection for telomerase activity is of great value in quantitative detection.

Example 9: Detection of the Telomerase Activity in Sputum Specimen from Lung Cancer Patient with the WATA Detection Kit for Telomerase Activity

(87) 20 lung cancer patients were in stage I as diagnosed by needle biopsy of tissues, and have not receive an operation. The sputum was freshly collected in the morning.

(88) The kit and operation steps were as described in Example 5.

(89) Among the 20 lung cancer patients, 16 were detected to be telomerase positive, and 4 were telomerase negative. 2 normal person (who had sputum due to smoking, and were healthy by recently physical examination) control were detected to be telomerase negative.

(90) TABLE-US-00025 Sample/Control Ct Results Negative control 29.68 Negative Positive control 20.84 Positive Lung cancer patient JFK-ZJL-201 16.33 Positive Lung cancer patient JFK-ZJL-202 19.58 Positive Lung cancer patient JFK-ZJL-203 26.54 Positive Lung cancer patient JFK-ZJL-204 18.29 Positive Lung cancer patient JFK-ZJL-205 30.43 Negative Lung cancer patient JFK-ZJL-206 31.76 Negative Lung cancer patient JFK-ZJL-207 24.99 Positive Lung cancer patient JFK-ZJL-208 27.86 Positive Lung cancer patient JFK-ZJL-209 19.19 Positive Lung cancer patient JFK-ZJL-210 16.93 Positive Lung cancer patient JFK-ZJL-211 17.40 Positive Lung cancer patient JFK-ZJL-212 26.62 Positive Lung cancer patient JFK-ZJL-213 21.52 Positive Lung cancer patient JFK-ZJL-214 28.74 Negative Lung cancer patient JFK-ZJL-215 18.47 Positive Lung cancer patient JFK-ZJL-216 20.78 Positive Lung cancer patient JFK-ZJL-217 29.85 Negative Lung cancer patient JFK-ZJL-218 27.22 Positive Lung cancer patient JFK-ZJL-219 26.59 Positive Lung cancer patient JFK-ZJL-220 20.55 Positive Normal person JFK-ZJN-001 28.96 Negative Normal person JFK-ZJN-002 29.75 Negative

(91) It can be seen that the telomerase activity in fresh sputum from most of the lung cancer patients can be detected by the WATA method.

Example 10: Detection of the Telomerase Activity in Surgically Removed Cervical Cancer Tissues with the WATA Detection Kit for Telomerase Activity

(92) The surgically removed cervical cancer tissues from 10 cervical cancer patients were frozen at 80 C. About 30 mg frozen cervical cancer tissue was clipped by sterilized ophthalmic scissors. 10 ml of the pretreatment buffer was added, and centrifuged. The supernatant was discarded. 200 l of the lysis buffer T was added to the pellet, and stood in an ice bath for 30 min. The following operations were as described in Example 5. The kit was as shown in Example 5.

(93) The cervical cancer tissues from the 10 cervical cancer patients are all telomerase positive as detected by WATA.

(94) TABLE-US-00026 Sample/Control Ct Results Negative control 29.77 Negative Positive control 20.53 Positive Cervical cancer patient JFK-ZJC-001 20.47 Positive Cervical cancer patient JFK-ZJC-002 15.62 Positive Cervical cancer patient JFK-ZJC-003 22.78 Positive Cervical cancer patient JFK-ZJC-004 19.91 Positive Cervical cancer patient JFK-ZJC-005 21.35 Negative Cervical cancer patient JFK-ZJC-006 24.88 Negative Cervical cancer patient JFK-ZJC-007 17.18 Positive Cervical cancer patient JFK-ZJC-008 20.26 Positive Cervical cancer patient JFK-ZJC-009 18.71 Positive Cervical cancer patient JFK-ZJC-010 17.55 Positive

(95) It can be seen that the telomerase activity in surgically removed frozen cancer tissues from the cervical cancer patients can be detected by the WATA method.

Example 11: Detection of the Telomerase Activity in ALT+ Lung Cancer Cell Line (SK-LU-1 Cells) with the WATA Detection Kit for Telomerase Activity

(96) ALT (alternative lengthening of telomere) is a mechanism by which the telomeric DNA length is extended and maintained in the telomerase negative cancer cells. SK-LU-1 (commercially available from ATCC, US), a known ALT.sup.+ cell, was cultured in a 24-well plate (at a density of about 10-10.sup.5 cells/well). The kit and operation steps were as described in Example 5. The lysate supernatant of 1000 SK-LU-1 cells was thermally deactivated as described in Example 3.

(97) The results are shown below.

(98) TABLE-US-00027 Sample/Control Ct Results 10 cells 29.71 Negative 100 cells 28.98 Negative .sup.10.sup.3 cells 29.18 Negative .sup.10.sup.4 cells 29.73 Negative .sup.10.sup.5 cells 29.24 Negative Negative control 29.68 Negative Positive control 20.11 Positive Thermally deactivated 29.93 Negative lysate supernatant

(99) The 10-10.sup.5 SK-LU-1 cells are all telomerase negative as detected by WATA, further confirming the specificity of the WATA method for detecting the telomerase activity.

Example 12: Effect of the WATA Method with a TaqMan Probe Substituting for SYBR Green I on Detection of the Telomerase Activity

(100) The primer and probe sequence, the reaction tube, the lysis buffer T, the reaction solution T, and the RE/PCR system were the same as those in Example 1. In the RE/PCR system, the SYBR green I was replaced by 0.5 mol/L TaqMan probe (having a sequence of 5-FAM-TAACCCTAACCCTAACCCTAACCCTAACCC-TAMRA-3). The cells were A549 cells. The detection method were as described in Example 3. The FAM fluorescent quantitative assay was used in place of the SYBR green I assay. When the Ct value was 30, a positive result was determined, and when the Ct value was >30, a negative result was determined.

(101) The result is shown below.

(102) TABLE-US-00028 Sample Ct Results Lysis buffer No Ct Negative 10 cells 29.82 Positive 100 cells 25.37 Positive 1000 cells 22.15 Positive 10000 cells 18.61 Positive Thermally deactivated 33.54 Negative lysate supernatant

(103) It can be seen that the telomerase activity in 10-10000 A549 cells can be detected by the WATA method using TaqMan.