Inspection method for improving positive rate of human papilloma virus (HPV) nucleic acid detection

Abstract

The invention discloses a detection method for improving the positive rate of human papilloma virus (HPV) nucleic acid detection, which is characterized in that on the premise of keeping the morphology and nucleic acid completeness of cervical exfoliated cells, red blood cells are completely dissolved, mucus is dispersed, CC points (monitoring hybridization process) and PC points (monitoring extraction and amplification process) of a sample are colored, the success rate of an experiment is ensured, and the detection accuracy is improved. According to comparison experiment results, the processed HPV nucleic acid detection experiment is high in success rate and convenient and simple to operate, the pain of resampling of a patient is reduced, the waste of repeated test reagents is avoided, the cost is saved, the generation of false negative results is avoided, and the accuracy of HPV nucleic acid detection results is improved, namely, the positive rate of the HPV nucleic acid detection results is improved.

Description

Technical Field

[0001] The invention relates to a method for detecting human papillomavirus (HPV) nucleic acid. Background Art

[0002] Human papillomavirus (HPV) belongs to the genus Papillomavirus of the family Papillomaviridae. It is a spherical, non-enveloped, double-stranded DNA virus with a genome length of 8000bp, icosahedral symmetry, no envelope, and a diameter of 45-55nm. Its nucleic acid is mainly divided into early coding region, late coding region and non-coding region according to its function. The late coding region is divided into L1 and L2, and the early coding region is divided into E1, E2, E4, E5, E6 and E7. It has functions such as viral replication, transcriptional regulation and cell transformation. E1 and E2 are both related to viral replication and are often used as viral integration sites. More than 200 species have been isolated so far. Infection mainly occurs in epithelial tissues, including the cervix, anus and oropharynx, and can cause proliferation of squamous epithelial cells in the skin and mucosa of the human body. Globally, cervical cancer is the third leading cause of cancer death in women, with approximately 528,000 new cases and approximately 266,000 deaths each year. According to statistics from 2016, the HPV positive rate in oropharyngeal squamous cell carcinoma (OPSCC) is 20.6%, in laryngeal cancer is 23.6%, in paranasal sinus cancer is 29.6%, and in nasopharyngeal cancer is 31.1%. Therefore, the detection rate of HPV is crucial for the prevention and treatment of cancers such as cervical cancer, oropharyngeal squamous cell carcinoma (OPSCC), laryngeal cancer, paranasal sinus cancer, and nasopharyngeal cancer. At present, the main HPV detection methods are gene amplification and molecular hybridization. Whether the pre-treatment of the specimen is correct and appropriate is related to the success or failure of the HPV detection experiment and is directly related to the detection rate of HPV. As we all know, bloody HPV test specimens account for 20-40% of the total number of specimens. Bloody cervical exfoliated cell specimens contain a certain amount of hemoglobin, which will inhibit the gene amplification reaction and cause false negative results; mucous HPV test specimens account for 10-20% of the total number of specimens. The mucus in the mucous specimens wraps the epithelial cells in the mucous substance. During the nucleic acid extraction process, the epithelial cells with HPV virus cannot precipitate, resulting in low nucleic acid extraction efficiency and failure of the gene amplification experiment. Summary of the invention

[0003] The purpose of the present invention is to provide a testing method for improving the positive rate of human papillomavirus (HPV) nucleic acid detection.

[0004] The technical solution of the present invention is: a test method for improving the positive rate of human papillomavirus (HPV) nucleic acid detection, characterized in that:

[0005] (1) Collecting cervical exfoliated cells and placing them in exfoliated cell preservation solution;

[0006] (2) Take the preservation solution containing the cervical exfoliated cell sample and place it in an EP tube.

[0007] (3) Add 0.15-0.25:1 of 3.5-5.8% sodium bicarbonate (NaHCO3) into the EP tube containing the exfoliated cell sample, mix thoroughly, and let stand;

[0008] (4) Take the EP tube containing the cervical exfoliated cell sample, centrifuge it at high speed, and remove the supernatant;

[0009] (5) Add 0.9% physiological saline, mix thoroughly to precipitate, and take the sample liquid containing the exfoliated cells for testing to obtain the test results.

[0010] The present invention has the following technical effects: the HPV typing nucleic acid detection specimen contains blood or (and) mucus cervical exfoliated cell specimen pretreatment inspection method of the present invention, under the premise of maintaining the morphology of cervical exfoliated cells and the integrity of nucleic acid, all red blood cells are dissolved and mucus is dispersed, and the CC point (monitoring hybridization process) and PC point (monitoring extraction and amplification process) of the sample are all colored, ensuring the success rate of the experiment, and by comparing the experimental results, it is concluded that the treated HPV nucleic acid detection experiment has a high success rate, is convenient and simple to operate, and avoids experimental inhibition caused by the sample containing blood; avoids the epithelial cells with HPV virus being wrapped in mucus substances due to the sample containing mucus, and the epithelial cells with HPV virus cannot be precipitated during the nucleic acid extraction process, resulting in low nucleic acid extraction efficiency. It reduces the pain of patients resampling, avoids the waste of repeated test reagents, saves costs, avoids the generation of false negative results, and improves the accuracy of HPV nucleic acid detection results, that is, improves the positive rate of HPV nucleic acid detection results. DETAILED DESCRIPTION

[0011] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the embodiments. The present invention uses the exfoliated cell preservation solution produced by Guangzhou Heshi Biotechnology Co., Ltd., and the detection instrument uses an integrated nucleic acid detector.

[0012] Example 1

[0013] Take 1 HPV specimen containing blood, and the processing method is as follows: mix the specimen → take 1000μl of the specimen and add it to a 1.5ml EP tube → add 200μl of 3.5-5.8% sodium bicarbonate (NaHCO3) to the EP tube → mix → let stand at room temperature for 10 minutes → high-speed centrifuge the EP tube at 12000rpm for 10min → remove 1150μl of the supernatant in the EP tube → add 250μl of 0.9% saline to the EP tube → mix thoroughly to precipitate → take 300μl of the specimen liquid for testing.

[0014] Example 2

[0015] Take 1 HPV specimen containing mucus, and the processing method is as follows: mix the specimen → take 1000μl of the specimen and add it to a 1.5ml EP tube → add 150μl of 3.5-5.8% sodium bicarbonate (NaHCO3) to the EP tube → mix → let stand at room temperature for 30 minutes → high-speed centrifuge the EP tube at 12000rpm for 10min → remove 850μl of the supernatant in the EP tube → mix the precipitate thoroughly → take 300μl of the specimen liquid for testing.

[0016] Example 3

[0017] Take 1 HPV specimen containing blood and mucus, and the processing method is as follows: mix the specimen → take 1000μl of the specimen and add it to a 1.5ml EP tube → add 250μl of 3.5-5.8% sodium bicarbonate (NaHCO3) to the EP tube → mix → let stand at room temperature for 30 minutes → high-speed centrifuge the EP tube at 12000rpm for 10min → remove 1200μl of the supernatant in the EP tube → add 250μl of 0.9% saline to the EP tube → mix thoroughly to precipitate → take 300μl of the specimen liquid for testing.

[0018] Comparison between existing detection methods and the present invention

[0019] From January to December 2023, a total of 3,630 HPV specimens were tested using the existing detection method, with a total of 692 positive tests and a positive rate of 19.06%; a total of 780 positive tests were detected using the present invention, with a positive rate of 21.48%, and the positive rate increased by 2.42 percentage points.

[0020] From January to September 2024, a total of 5798 HPV specimens were tested using the existing detection method, with a total of 1101 positive tests and a positive rate of 18.98%; using the present invention, a total of 1218 positive tests were conducted, with a positive rate of 21.00%. The positive rate increased by 2.02 percentage points.

Claims

1. A method for improving the positive rate of human papillomavirus (HPV) nucleic acid detection, characterized in that: (1) Collecting cervical exfoliated cells and placing them in exfoliated cell preservation solution; (2) Take the preservation solution containing the cervical exfoliated cell sample and place it in an EP tube. (3) Add 0.15-0.25:1 of 3.5-5.8% sodium bicarbonate (NaHCO3) into the EP tube containing the exfoliated cell sample, mix thoroughly, and let stand; (4) Take the EP tube containing the cervical exfoliated cell sample, centrifuge at high speed, and remove the supernatant; (5) Add 0.9% physiological saline, mix thoroughly to precipitate, and take the sample liquid containing the exfoliated cells for testing to obtain the test results.