Cervical cancer diagnosing method and diagnostic kit for same
View Patent ↗There are provided an improved cervical cancer diagnosing method and a diagnostic kit for same. According to the present invention, it is possible to more rapidly and accurately provide a patient group requiring a clinical treatment and a prevention treatment in terms of a technical aspect to predict that the limitation of the existing HPV DNA test method can be overcome, automate the RNA extraction from eliminated cells, and more rapidly provide more objective and accurate results because the result analysis can be performed by software by using the real-time RT-PCR.
1. A method for increasing the likelihood of making a positive cervical cancer diagnosis, comprising:
a) separating a total RNA from cells obtained from a specimen;
b) synthesizing cDNA from a separated total RNA;
c) performing a multiplexed real-time-PCR for the synthesized cDNA by using a primer set and a detection probe capable of amplifying and detecting HPV high-risk types 16, 35, 31, 58, 33, and 52; a primer set and a detection probe capable of amplifying and detecting HPV high-risk types 18, 45, 39, 68, and 59; a primer set and a detection probe capable of amplifying and detecting HPV high-risk types 53, 56, 66, 51, 69, 26, and 30; a primer set and a detection probe capable of amplifying and detecting glvceraldehvde 3-phosphate dehydrogenase (GAPDH) as a reference gene; and a primer set and a detection probe capable of amplifying and detecting the human telomerase reverse transcriptase (hTERT) gene as a prognosis marker for the cervical cancer;
d) detecting an expression level of an amplified HPV high-risk type and quantifying the expression level of the amplified HPV high-risk type based on an expression level of GAPDH; and
e) comparing an expression level of the hTERT gene in the specimen with an expression level of the hTERT gene in a normal person after quantifying the expression level of the hTERT gene in the specimen based on the expression level of GAPDH,
wherein an expression level of the hTERT gene in the specimen that is higher than the expression level of the hTERT gene in a normal person increases the likelihood that a specimen also having a detected expression level of an amplified HPV high-risk type is indicative of a positive cervical cancer diagnosis.
2. The method of claim 1 , wherein the primer set capable of amplifying the HPV high-risk types 16, 35, 31, 58, 33, and 52 are represented by SEQ ID NOS: 1 and 2 and the detection probe has a base sequence represented by SEQ ID NO: 3.
3. The method of claim 1 , wherein the primer set capable of amplifying the HPV high-risk types 18, 45, 39, 68, and 59 are represented by SEQ ID NOS: 4, 5, 6 and 7 and the detection probe has a base sequence represented by SEQ ID NO: 8.
4. The method of claim 1 , wherein the primer set capable of amplifying the HPV high-risk types 53, 56, 66, 51, 69, 26, and 30 are represented by SEQ ID NOS: 9 and 10 or 12 and 13 and the detection probe has a base sequence represented by SEQ ID NO: 11 or 14.
5. The method of claim 1 , wherein the primer set capable of amplifying the GAPDH gene are represented by SEQ ID NOS: 15 and 16 and the detection probe has a base sequence represented by SEQ ID NO: 17, and the primer set capable of amplifying the hTERT gene are represented by SEQ ID NOS: 18 and 19 and the detection probe has a base sequence represented by SEQ ID NO: 20.