Kit for rapidly detecting human papilloma virus gene

Abstract

The invention relates to the technical field of gene detection kits, in particular to a kit for rapidly detecting human papilloma virus genes, which comprises reagent bottles or tubes containing nucleic acid release liquid, enzyme mixed liquid, primer mixed liquid, a negative quality control product and a positive quality control product, and a packaging box for separating and intensively packaging the reagent bottles or tubes, the kit adopts an LAMP amplification system without a probe, a detection result of the human papilloma virus can be obtained within 10-15 minutes through a fluorescent dye and interpretation equipment, and the kit has the characteristics of high sensitivity, high specificity, simplicity in operation and high detection speed, and can be widely applied to auxiliary diagnosis of the human papilloma virus.

Description

Technical Field

[0001] The present application relates to the technical field of gene detection kits, and in particular to a kit for rapid detection of human papillomavirus genes; the kit can be widely used in auxiliary diagnosis of human papillomavirus by performing qualitative detection of human papillomavirus in a sample. Background Art

[0002] In the existing technology, isothermal amplification is a nucleic acid amplification technology that is currently widely used in scientific research and clinical testing. It amplifies the amount of trace DNA or RNA templates by 100,000 to 1 million times. By adding fluorescent dyes during the amplification process, the product increase process can be graphically depicted in real time, so it is often used to observe the presence or amount of genes. Among the many isothermal amplification technologies, the most reliable and most commonly used is LAMP (loop-mediated isothermal amplification). Compared with the currently widely used PCR technology, it has the following advantages: (1) It is fast, with the entire amplification process completed in 10-15 minutes, and the amplification multiple is comparable to PCR, which has obvious advantages in scenarios that require timeliness, such as emergency treatment; (2) The requirements for template purity are looser than those of the PCR system, and it is compatible with relatively rough lysis methods; (3) It has better specificity and a lower commercialization cost than PCR.

[0003] Human papillomavirus is a group of spherical, tiny, non-enveloped circular double-stranded DNA viruses belonging to the genus Papillomavirus of the family Papovaviridae. It is epithelial and mainly causes proliferative lesions of human skin and mucous membranes. It is divided into three categories: high-risk, medium-risk and low-risk. The symptoms it can cause include common warts, genital warts (condyloma acuminatum), cervical cancer, anal cancer, etc. The methods currently used in clinical testing and scientific research mainly include fluorescent quantitative PCR technology (CN104818342A), chip hybridization technology (CN101818213A) and high-throughput sequencing methods (CN101871014A). These technologies have been used in the field of pathogen detection for many years and are relatively mature, but there are certain shortcomings in timeliness and usage scenarios.

[0004] In recent years, the timeliness requirements for disease detection have become increasingly stringent. Although it has not yet been widely used, LAMP technology does overcome the main shortcomings of COVID-19 detection: (1) It takes too long to detect a single sample; (2) The pre-treatment requirements for samples are relatively stringent; (3) Cost constraints lead to mixed testing of samples, resulting in reduced sensitivity.

[0005] In the LAMP amplification system, it only requires 4-6 primers and no probes. Through fluorescent dyes and reading equipment, a positive amplification signal can be obtained within 10-15 minutes. If no signal is generated during the 15-minute amplification process, the sample is judged to be negative. Its performance is no less than PCR, but its cost is significantly lower than PCR. Summary of the invention

[0006] The purpose of the present invention is to provide a kit for rapid detection of human papillomavirus genes, comprising reagent bottles or tubes containing nucleic acid release solution, enzyme mixture, primer mixture, negative quality control product, and positive quality control product, and a packaging box for separating and centrally packaging these reagent bottles or tubes; the kit adopts a LAMP amplification system without a probe, and can obtain the detection result of human papillomavirus within 10-15 minutes through fluorescent dyes and interpretation equipment, and has the characteristics of high sensitivity, high specificity, simple operation and fast detection speed.

[0007] In order to solve the above tasks, the specific technical route of the present invention is: (1) Three pairs of primers were designed for the conserved sequences of human papillomavirus, named P 左外 With P 右外 , P left center and P 右中 , P 左内 With P 右内 ; (2) preparing a reaction system suitable for LAMP amplification. The reaction system includes Bst polymerase, reverse transcriptase, divalent magnesium ions, fluorescent dye, and a buffer solution composed of a mixture of deoxyribonucleotides of guanine, adenine, thymine, and cytosine; (3) DNA or RNA is extracted from the sample to be tested, added to the aforementioned reaction system, amplified at 65°C for 15 minutes, and a fluorescence value is read every 20 seconds or 30 seconds to form a curve to determine whether the HPV gene exists.

[0008] According to an embodiment of the present invention, the enzyme mixture suitable for the LAMP amplification reaction system is composed of Bst polymerase and reverse transcriptase, and the primer mixture is composed of a buffer, divalent magnesium ions, a fluorescent dye and three pairs of primers, characterized in that the sequences of the three pairs of primers used for target polynucleotide amplification are respectively, The left outer portion of P is 5'-GACACGGTATCCGCTACTCAG-3' (SEQ ID NO: 1), P right outside is 5'-CTGTAGCTGCACCGAGAAGTG-3' (SEQ ID NO: 2), P left middle is 5'-GGACACGGTGCTGGAATACG-3' (SEQ ID NO: 3), P right middle is 5'-GACCTGGACACTGTGGACTCG-3' (SEQ ID NO: 4), The inner left side of P is 5'-CCGTAGGTCTTTTTGGTGCCCAGCTACAGCACACCCCCTCA-3' (SEQ IDNO: 5), In the right side of P is 5'-CCAGACGTCGGCTGCTACACGGTTGACAGGTCCACAATGC-3' (SEQ ID NO: 6).

[0009] Preferably, P 左外 With P 右外 The final concentration in the reaction system is 0.1-0.2uM, P 左中 With P 右中 The final concentration in the reaction system is 0.3-0.9uM, P 左内 With P 右内 The final concentration in the reaction system is 1.0-1.8uM.

[0010] Preferably, Bst polymerase is a hot start enzyme that can be stored at room temperature.

[0011] Compared with the prior art, the present invention has the following advantages: (1) The LAMP amplification system is used, which requires fewer primers and no probes; (2) Amplification positive signals can be obtained within 10-15 minutes through fluorescent dyes and interpretation equipment; (3) The cost of using the LAMP amplification system is lower than that of the PCR system. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Temperature program setting diagram for the human papillomavirus detection kit (Hongshi SLAN-96S).

[0013] Figure 2 This is the fluorescence amplification curve of positive samples from the human papillomavirus detection kit.

[0014] Figure 3 This is the amplification curve of the positive quality control product of the human papillomavirus detection kit.

[0015] Figure 4 This is the fluorescence amplification curve of the negative control product of the human papillomavirus detection kit. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0017] Example 1: Detection method of human papillomavirus gene kit (1) Sample collection, transportation and storage Place the specimens to be tested and the vaginal swabs after sampling into a dry special small box. They can be sent for testing directly or stored in a 4°C refrigerator. The storage period should not exceed one month. If necessary, transport them at room temperature.

[0018] (2) Sample processing a) Place the swab in a centrifuge tube containing 300 μl Trizol and 100 μl chloroform, break the swab, cover the tube, shake vigorously for 20 seconds, let stand for 3 minutes, and then centrifuge at 12,000 rpm for 2 minutes; b) Carefully remove the colorless upper layer of liquid (be careful not to touch the middle flocculent layer), transfer it to a newly sterilized 1.5 ml centrifuge tube, then add 10 μl of glass milk (mix thoroughly and then aspirate), mix thoroughly with an oscillator, centrifuge at 8000 rpm for 1 minute, and carefully discard all the liquid; c) Add 400 μl of 75% DEPC ethanol, shake thoroughly to mix, centrifuge at 8000 rpm for 1 minute, and drain the liquid as much as possible (use the pipette tip to drain the residual liquid to avoid touching the precipitated particles); d) Place the centrifuge tube containing the precipitate in a fume hood to air-dry for 15 minutes (you can also use an open heater to dry at 60°C for 5 minutes to prevent cross-contamination of samples), then add 30μl of nuclease-free H2O, fully resuspend with an appropriate pipette, let stand for 1 minute, and then centrifuge at 8000rpm for 1 minute. The processed samples can be directly used for PCR detection or temporarily stored at -20°C. If stored for more than one month, they can be stored at -70°C.

[0019] (3) LAMP amplification detection a) Preparation system Mix the primer-probe mixture and enzyme mixture thoroughly at a ratio of 2:1, and then dispense 15 μl into each 0.2 μl reaction tube; b) Adding samples Add 10 μl of the extract of the sample to be tested (or negative or positive quality control) into the reaction tube, cover the tube tightly, centrifuge it instantly, and then put it into the thermal amplification instrument for amplification.

[0020] (4) Amplification program settings I. The parameters of ABI7300 / 7500 fluorescence PCR amplification instrument are set as follows: Reporter Dye: FAM; QuencherDye:none; PassiveReference: none.

[0021] The amplification temperature parameters were uniformly set as follows: 65°C, 30 seconds, read the fluorescence value, 30 cycles; II. The parameters of the Hongshi SLAN-96S fluorescence PCR amplification instrument are set as follows: Use the first channel, 65°C, 20 seconds, read the fluorescence value, and repeat for 45 cycles.

[0022] (5) Result determination If there is no S-shaped curve in the amplification, the sample is judged to be negative; If the amplification curve shows an obvious S-shape, it is judged as a positive sample.

[0023] Example 2: Use of quality control products in human papillomavirus gene kit The quality control products in the human papillomavirus nucleic acid detection kit include positive quality control products and negative quality control products, which are used for quality control in clinical trials, and the operation method is the same as that of the samples to be tested.

[0024] The quality control standards are as follows: The amplification curve of the negative control product showed no obvious S-shaped growth; The amplification curve of the positive quality control product was S-shaped.

[0025] It is to be understood that the present application is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present application. In addition, under the guidance of the present application, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present application. Therefore, the present application is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of the present application are within the scope protected by the present application.

Claims

1. A kit for rapid detection of human papillomavirus genes, comprising reagent bottles or tubes containing nucleic acid release solution, enzyme mixture, primer mixture, negative quality control product, and positive quality control product, and a packaging box for separating and centrally packaging these reagent bottles or tubes, wherein the enzyme mixture suitable for LAMP amplification reaction system is composed of Bst polymerase and reverse transcriptase, and the primer mixture is composed of buffer, divalent magnesium ions, fluorescent dye and three pairs of primers, characterized in that: The sequences of the three pairs of primers used for target polynucleotide amplification are: P 左外 is 5’-GACACGGTATCCGCTACTCAG-3’, P 右外 is 5'-CTGTAGCTGCACCGAGAAGTG-3', P 左中 is 5’-GGACACGGTGCTGGAATACG-3’, P 右中 is 5’-GACCTGGACACTGTGGACTCG-3’, P 左内 is 5’-CCGTAGGTCTTTTTGGTGCCCAGCTACAGCACACCCCCTCA-3’, P 右内 is 5’-CCAGACGTCGGCTGCTACACGGTTGACAGGTCCACAATGC-3’.

2. The kit according to claim 1, characterized in that: The enzyme mix contains an RNase inhibitor.

3. The kit according to claim 1, characterized in that: Reverse transcriptase comes from birds.

4. The kit according to claim 1, characterized in that: The primer mix contains a small molecule, fluorescent dye with a wavelength of 500-530nm.

5. The kit according to claim 1, characterized in that: The buffer is a mixture of deoxyribonucleotides of guanine, adenine, thymine and cytosine.

6. The kit according to claim 1, characterized in that: P 左外 With P 右外 The final concentration in the reaction system is 0.1-0.2uM.

7. The kit according to claim 1, characterized in that: P 左中 With P 右中 The final concentration in the reaction system is 0.3-0.9uM.

8. The kit according to claim 1, characterized in that: P 左内 With P 右内 The final concentration in the reaction system is 1.0-1.8uM.

9. The kit according to claim 1, characterized in that: Bst polymerase is a hot-start enzyme that can be stored at room temperature.

10. The kit according to claim 1, characterized in that: The negative quality control is pure water treated with diethylene glycol pyrocarbonate (DEPC), and the positive quality control is a plasmid containing a specific human papillomavirus fragment.

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