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HPV detection kit

A detection kit, the technology of the kit, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, microorganisms, etc., can solve the problem that clinical diagnosis cannot be widely used, the detection cost is as high as 500 yuan, and the screening cannot be widely used. and other problems, to achieve the effect of low typing technology cost, reduced inspection cost and high specificity

Active Publication Date: 2015-04-29
镇江爱必梦生物科技有限公司
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  • Abstract
  • Description
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Problems solved by technology

In addition, depending on the matching positions of these few bases, sometimes the DNA polymerase used in the qPCR reaction system can further amplify the formed primer-dimer, so that sometimes the CT value of the primer-dimer It can be as low as about 25, which seriously interferes with the interpretation of the experimental results
For a long time, double-stranded DNA combined with fluorescent dye qPCR method is cheap, but due to the above reasons, it cannot be widely used in clinical diagnosis.
[0008] Probe qPCR can effectively solve the non-specific problem of double-stranded DNA binding fluorescent dye qPCR, but its main disadvantage is that the cost of probe design is too high to be widely used in the screening of diseases such as HPV infection.
The reason is that more than 50 clinically significant HPV types have been discovered, and more than 20 high-risk HPV subtypes require the design of more than 20 specific Taqman probes for detection, so that the detection cost of a sample is often as high as 500 diverse

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Primer design.

[0050] Use Vector NTi bioinformatics software to systematically analyze the biological information of various (high-risk and low-risk) HPV genomes, and select the conserved HPV L1 gene fragments in the HPV genome that are not homologous to human and other microbial genome DNA sequences as targets. Primers capable of specifically amplifying all types of HPV L1 gene fragments are designed, and the sequences of the obtained primers are shown in SEQ ID NO: 1-2.

[0051] SEQ ID NO: 1 (F1): 5'---GCCATTAGGTGTGGGCATTAGTGG---3';

[0052] SEQ ID NO: 2 (R1): 5'---TCCTTTGCCCCAGTGTTCCCC---3'.

[0053] The conserved sequences of E6 genes of HPV 16, 31, 33, 35, 51, 58, 59, and 82 types were analyzed by Vector NTi bioinformatics software, and a design that can specifically amplify 16, 31, 33, 35, Primers for 51, 58, 59, and 82 types of high-risk HPV, and the obtained primer sequences are shown in SEQ ID NO: 3-4.

[0054] SEQ ID NO: 3 (F2): 5'---CCGTTGTGTG...

Embodiment 2

[0075] Example 2: Experimental design for HPV detection using the primers obtained in Example 1.

[0076] In this example, HPV molecular detection is completed by 8-tube qPCR. The specific experimental design is as follows: figure 2 shown. Tube 1 is used to detect all types of HPV, including various high-risk and low-risk types; tubes 2 to 7 detect all types of high-risk HPV; tube 8 is the quality control reaction of the β-actin gene internal reference, and the specific functions of tubes 1 to 8 are analyzed as follows :

[0077] (1) Tube 1 uses the primer pair of SEQ ID NO: 1-2. If the reaction of tube 1 is positive, it means that the patient has HPV virus infection, which may be high-risk or low-risk HPV type.

[0078] (2) Tube 2 uses the primer pair of SEQ ID NO: 3-4. If the reaction of tube 2 is positive, it means that the patient has high-risk HPV virus infection, which may be 16, 31, 33, 35, 51, 58, 59 or 82 types; Type 16 accounts for more than 60% of all high-risk ...

Embodiment 3

[0085] Embodiment 3: establishment of standard curve.

[0086] In order to test whether the designed primers can be accurately applied to the qPCR molecular detection of HPV, the siHA cell line known to contain HPV16 was used to detect the standard curve of the primer pair of SEQ ID NO:3-4. A Hela cell line known to contain HPV18 was used to detect the standard curve of the primer pair of SEQ ID NO:5-6.

[0087] The siHA containing HPV16 type and the Hela (cell genomic DNA) containing HPV18 type were respectively extracted with a genomic DNA extraction kit (Kangwei Century Biotechnology, Beijing), and then the genomic DNA was diluted to 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 individual / group. Then use tubes 2 and 3 primer pairs to detect them respectively according to the qPCR reaction system and reaction conditions in Table 1 and Table 2. The results showed that the qPCR CT value was negatively correlated with the amount of genomic DNA, and the reproducibility was...

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Abstract

The invention discloses a qPCR primer for detecting HPVs. The qPCR primer comprises the following primer pairs of two types: the primer pairs of the first type are capable of amplifying conserved sequences in the HPV genomes of all the types; the primer pairs of the second type are capable of detecting the HPVs of at least two types in the same tubular qPCR system. The invention also discloses an HPV detection kit comprising the primer pairs. The HPV detection kit is high in HPV detection specificity and completely reaches the specificity level of qPCR by use of a probe method. Compared with traditional PCR diagnosis, the HPV detection kit is simpler and more reliable to operate, and meanwhile, applicable to high-throughput sample detection. The HPV detection kit further has the characteristics of high specificity, strong sensitivity and low cost by applying a double-stranded DNA combined fluorochrome qPCR detection method to the diagnosis of the HPVs; as a result, a quick and accurate molecular detection method is provided for general investigation of the HPVs and prevention and treatment of the cervical cancer, and meanwhile, the test cost is greatly reduced; in short, the HPV detection kit has an important popularization and application value.

Description

technical field [0001] The invention belongs to the technical field of human papillomavirus (HPV) screening, and in particular relates to an HPV detection kit and a detection method thereof. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) is an epitheliophilic virus with a high degree of specificity, and humans are its only host. HPV has a special tropism for the epidermis and mucosal squamous epithelium. Its genome is double-stranded circular DNA, containing about 7900 base pairs (bp), and consists of three gene regions, including the early region (Early Region, E region), Late region (Late Region, L region) region and non-coding region (Uncoding Region, UCR) or upstream regulatory region (URR). The E region consists of seven genes in sequence: E6, E7, E1, E2, E3, E4, and E5, which are involved in the replication of viral DNA, transcription, encoding viral proteins, and maintaining a high copy number of intracellular viruses. Among them, E6 a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/708C12Q2600/112C12Q2563/107
Inventor 李沛祥李军王倩高源远刘德斌印思源
Owner 镇江爱必梦生物科技有限公司
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