A primer composition for detecting different types of human papilloma virus and application thereof

Abstract

The application provides a primer composition for detecting different types of human papilloma virus and application thereof, and relates to the technical field of nucleic acid determination or detection methods.The primer composition provided by the application comprises a first primer and a second primer for detecting HPV type 16, a third primer and a fourth primer for detecting HPV type 18, and a fifth primer and a sixth primer for detecting HPV high-risk types.The primer composition provided by the application can realize multiplex detection of human papilloma virus, has the characteristics of good specificity, high sensitivity and good repeatability, and can avoid the false positive problem existing in conventional primers.Based on the primer composition, the application further combines an immunochromatography test strip detection technology to realize rapid and efficient detection of human papilloma virus, and the operation is simple and relatively portable, and the application can be used for home self-detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nucleic acid detection methods, and particularly relates to a primer composition for detecting different types of human papilloma virus and application thereof. BACKGROUND

[0002] Human papilloma virus (HPV) is widely present in nature, and humans are its first host. It is resistant to dryness and can be stored for a long period of time. At present, more than 150 subtypes of human papilloma virus have been isolated and identified. According to the size of pathogenicity or the size of carcinogenic risk, human papilloma virus can be divided into two categories: low-risk type and high-risk type. Human papilloma virus mainly infects skin and mucosal epithelial cells of humans. The low-risk type of skin is related to common warts, flat warts, plantar warts, etc., including human papilloma virus 1, 2, 3, 4, 7, 10, 12, 15, etc. The high-risk type of skin is related to warty dyskeratosis, and other possible cancers include vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer, etc., including human papilloma virus 5, 8, 14, 17, 20, 36, 38, etc. The low-risk type of mucosa mainly infects the genital, anal, oropharyngeal and esophageal mucosa, including human papilloma virus 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, etc. The high-risk type of mucosa mainly causes cervical cancer, rectal cancer, oral cancer, tonsil cancer, etc., including human papilloma virus 16, 18, 30, 31, 33, 35, 39.

[0003] Human papilloma virus infection is mainly transmitted through sex. When HPV contacts the skin and mucosa of the genital area, it is easy to cause viral infection, and then the epithelial cells will undergo a series of abnormal changes, including poor differentiation, disordered arrangement, large and darkly stained nuclei, and abnormal proliferation. Under normal circumstances, epithelial cells will differentiate and proliferate according to a certain program to maintain the normal structure and function of the tissue. In the early stage of the lesion, the infection of HPV interferes with this normal process, leading to abnormal cell morphology and function. In addition to sexual transmission, HPV can also be transmitted through contact and mother-to-child transmission, and the transmission route is extensive. Therefore, timely and rapid detection is of great significance and necessity.

[0004] At present, the main means for detecting HPV is polymerase chain reaction (PCR) technology, which is a technology for amplifying specific DNA fragments in vitro. This technology can be widely used in genetic diagnosis, infectious disease monitoring and other fields, but at the same time, the equipment used by this technology is large in size, inconvenient to operate, and the amplification reaction time is long, generally several hours, which is difficult to popularize and apply in the grass-roots level. In order to improve the portability and rapidity of HPV virus detection, the constant temperature PCR is a constant temperature nucleic acid rapid amplification technology, that is, under constant temperature, the polymerase in the reaction system uses specific primers for rapid nucleic acid amplification reaction. With the assistance of endonuclease, the modified specific molecular probe is amplified with the modified downstream primer to form a double-end modified nucleic acid fragment. With the accumulation of amplification products, the final result can be judged by using nucleic acid test paper for color development or using a small fluorescence detector for fluorescence result judgment. This method has the advantages of simple operation, rapid detection, simple operation, high detection sensitivity, etc. Therefore, it can realize home detection and rapid detection in the field.

[0005] Loop-mediated isothermal amplification (LAMP) is a common isothermal amplification technology, which has the advantages of high amplification efficiency, short reaction time and strong specificity. However, LAMP technology usually uses 4-6 primers to recognize multiple regions of the target sequence, which is complex in design. If the primer design is not proper or has some similarity with non-target DNA, it may cause non-specific binding and cause amplification of non-target sequences, resulting in false positive results. In addition, LAMP amplification products are very numerous, and conventional techniques can be used to cut the specific enzyme cutting site contained in the amplification product and perform electrophoresis detection, but the disadvantage of electrophoresis detection is that the amplification tube needs to be opened, and high-concentration DNA products are easy to produce aerosol and cause laboratory environment pollution; if there are residual products from previous amplification in the laboratory environment, it may contaminate the next reaction, resulting in false positives. Therefore, how to design primers for HPV based on the principle of isothermal amplification to improve the detection accuracy is one of the technical problems to be solved in the field. SUMMARY

[0006] The present application provides a primer composition for detecting human papillomavirus and its application, which solves the problem of high false positive rate in HPV detection.

[0007] In a first aspect, the present application provides a primer composition for detecting human papillomavirus, comprising a first primer, a second primer, a third primer, a fourth primer, a fifth primer and a sixth primer, wherein:

[0008] The structure general formula of the first primer, the second primer, the third primer, the fourth primer, the fifth primer and the sixth primer is shown as formula 1:

[0009] 5'-Y-n(a)-n(b)-n(c)-3' formula 1;

[0010] In formula 1, Y represents a labeling group, n(a) and n(c) represent polynucleotide fragments of different human papillomavirus genotypes, respectively, and n(b) represents a connecting sequence.

[0011] In the first primer, n(a) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 3, and n(c) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 4.

[0012] In the second primer, n(a) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 5, and n(c) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 6.

[0013] In the third primer, n(a) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 7, and n(c) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 8.

[0014] In the fourth primer, n(a) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 9, and n(c) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 10.

[0015] In the fifth primer, n(a) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 11, and n(c) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 12.

[0016] In the sixth primer, n(a) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 13, and n(c) represents a polynucleotide fragment with a nucleotide sequence of SEQ ID NO: 14.

[0017] The composition as described above, wherein the labeling group is independently selected from at least one of a fluorescent group, biotin, and digoxin. Further, the fluorescent group is selected from at least one of FITC, TAMRA, FAM, CY3, CY5, and Texas Red.

[0018] Further, the labeling group of the first primer, the third primer, and the fifth primer is preferably a fluorescent group, which facilitates tracking and detecting the presence of HPV genes in experiments. The labeling groups of the first primer, the third primer, and the fifth primer can be the same or different. The labeling groups of the second primer, the fourth primer, and the sixth primer are different from each other and are used to capture the amplification products corresponding to different primer pairs.

[0019] The composition as described above, wherein the connecting sequence is independently selected from one of TT, TTT, TTTT, TTTTT, and TTTTTT.

[0020] In a specific embodiment, the first primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 15 modified with a fluorescent group FAM at the 5' end; the second primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 16 modified with biotin at the 5' end; the third primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 17 modified with a fluorescent group FAM at the 5' end; the fourth primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 18 modified with digoxin at the 5' end; the fifth primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 19 modified with a fluorescent group FAM at the 5' end; and the sixth primer is a single-stranded DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 20 modified with a fluorescent group FITC at the 5' end.

[0021] The present application is based on isothermal amplification technology with different melting temperatures, belongs to improved LAMP, combines the primer design strategy of PCR and the constant temperature amplification advantage of LAMP. The genomic sequences of different subtypes of human papillomavirus are selected, and the target sequence melting temperature (Tm) has a significant change in the nucleic acid region. A pair of primers (D and P) are designed, so that their melting temperature is higher than the reaction temperature, and the local region of the target sequence has a lower melting temperature. At a constant temperature, the primers can efficiently anneal to the partially melted target sequence region and initiate a chain reaction. The amplification does not depend on thermal denaturation or auxiliary enzymes, and only Bst DNA polymerase is required. Because the melting temperature of the template has high and low changes, only the specific target sequence can be opened and recognized at this temperature, which improves the specificity and reduces false positives.

[0022] In a second aspect, the present application provides the use of the primer composition or the product described above in the preparation of a product for detecting human papillomavirus.

[0023] In a third aspect, the present application provides a method for detecting whether a sample contains human papillomavirus for non-diagnostic purposes, comprising:

[0024] Extracting DNA from the sample to be detected to obtain a DNA sample to be detected;

[0025] Using any of the primer compositions described above to perform an amplification reaction on the DNA sample to be detected to obtain an amplification product;

[0026] Detecting the amplification product, and determining whether the sample to be detected contains human papillomavirus according to the detection result.

[0027] The method described above, the sample to be detected can be blood, secretion, etc. Further, the secretion can be human cervical secretion.

[0028] The method as described above, wherein the human papillomavirus is a high-risk HPV, and specifically includes at least one of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68.

[0029] The method as described above, wherein the amplification reaction is performed at a constant temperature of 55-65℃.

[0030] The method as described above, wherein the DNA polymerase used in the amplification reaction is a Bst polymerase, such as Bst 2.0, Bst 3.0, etc. Further, the DNA polymerase is Bst 2.0.

[0031] The method as described above, wherein the amplification product is added dropwise to the sample area of the colloidal gold immunochromatography test strip, and whether the human papillomavirus is contained in the sample to be detected is determined by whether the corresponding band is colored.

[0032] In this document, the term "non-diagnostic purpose" refers to not aiming to obtain information about whether an individual is infected with the above-mentioned pathogen and suffers from the corresponding disease, i.e., not directly aiming to obtain a disease diagnosis result or health status.

[0033] In a fourth aspect, the present application also provides a portable detection device based on constant-temperature PCR reaction, comprising:

[0034] A box body is provided with a colloidal gold test paper inside, the box body is provided with an observation window, and the observation window is located above the colloidal gold test paper; the top of the box body is provided with a liquid outlet, and the upper surface of the box body is provided with a pipe seat, and the liquid outlet is located in the pipe seat;

[0035] A constant-temperature heating element is arranged on the top surface inside the box body and below the pipe seat;

[0036] A first stepped pipe cylinder is connected to the lower part of the pipe seat, and the bottom surface of the first stepped pipe cylinder is provided with a reaction chamber penetrating up and down;

[0037] A second stepped pipe cylinder is connected to the lower part of the first stepped pipe cylinder, and the bottom surface of the second stepped pipe cylinder is provided with a liquid inlet;

[0038] An end cover is buckled on the top of the second stepped pipe cylinder, and the end cover is provided with a droplet tube, and the bottom end of the droplet tube is located in the liquid inlet;

[0039] A sealing plug is plugged at the droplet opening of the droplet tube.

[0040] The detection device as described above, wherein one side of the box body is provided with a TYPE-C interface, and the TYPE-C interface is connected with the constant-temperature heating element.

[0041] The detection device described above includes an upper box and a lower box that are connected to each other.

[0042] As described above, in the detection device, a first slide rail is provided on the side wall of the tube seat, and a cylindrical first slider is provided on the first stepped tube, which can slide along the first slide rail.

[0043] As described above, the detection device has first limiting bosses at both ends of the first slide rail to limit the position of the first slider within the first slide rail.

[0044] As described above, in the detection device, a second slide rail is provided on the side wall of the first stepped tube, and a cylindrical second slider is provided on the second stepped tube, which can slide along the second slide rail.

[0045] As described above, the detection device has second limiting bosses at both ends of the second slide rail, which are used to limit the position of the second slider within the second slide rail.

[0046] In the detection device described above, a transparent plastic film is provided on the observation window.

[0047] In the detection device described above, the inner wall of the reaction chamber is configured as a rack shape.

[0048] As described above, the bottom surface of the second stepped tube is provided with a sealing boss, which is used to cover the top of the reaction chamber after the second stepped tube is rotated.

[0049] Fifthly, the present invention provides a product comprising any of the primer compositions described above.

[0050] The product described above may further include at least one of the portable detection device and the DNA polymerase required for the reaction.

[0051] The product described above can be a virus detection kit.

[0052] The primer composition provided by this invention enables multiplex detection of human papillomavirus (HPV), exhibiting high specificity, high sensitivity, and good repeatability, while avoiding the false-positive problem associated with conventional primers. Based on the above primer composition, this invention further combines immunochromatographic test strip detection technology to achieve rapid and efficient detection of HPV. The procedure is simple and portable, making it suitable for home self-testing. Attached Figure Description

[0053] Figure 1 This is a physical diagram of an HPV detection device according to an embodiment of the present invention; wherein, the left diagram is a left view and the right diagram is a top view;

[0054] Figure 2 is a structural schematic diagram of a portable detection device based on constant temperature PCR reaction provided by an embodiment of the present application;

[0055] Figure 3 is an exploded schematic diagram of the portable detection device based on constant temperature PCR reaction provided by the embodiment of the present application;

[0056] Figure 4 is a longitudinal sectional schematic diagram of the portable detection device based on constant temperature PCR reaction provided by the embodiment of the present application;

[0057] Figure 5 is a schematic diagram of an upper box body provided by the embodiment of the present application;

[0058] Figure 6 is a schematic diagram of a lower box body provided by the embodiment of the present application;

[0059] Figure 7 is a top view schematic diagram of a first stepped tube provided by the embodiment of the present application;

[0060] Figure 8 is a bottom view schematic diagram of the first stepped tube provided by the embodiment of the present application;

[0061] Figure 9 is a top view schematic diagram of a second stepped tube provided by the embodiment of the present application;

[0062] Figure 10 is a bottom view schematic diagram of the second stepped tube provided by the embodiment of the present application;

[0063] Figure 11 is a freeze-dried microsphere actual object diagram provided by an embodiment of the present application;

[0064] Figure 12 is a constant temperature nucleic acid detection result determination method of the present application; from left to right, when the sample does not contain HPV virus, only the C line shows red, T1-T3 is colorless, and it is determined as HPV negative; when the sample contains other high-risk viruses except HPV-16 and 18, the C line and the T1 line are red, T2-T3 is colorless, and it is determined as HPV high-risk positive; when the sample contains HPV-16 virus, the T1 line, the T2 line and the C line are all red at this time, and it is determined as HPV-16 high-risk positive; when the sample contains HPV-18 virus, the T1 line, the T3 line and the C line are all red at this time, and it is determined as HPV-18 high-risk positive infection result; when the sample contains HPV-16 and 18 high-risk viruses or more than two kinds of viruses, the T1 line, the T2 line, the T3 line and the C line are all red at this time, and it is determined as HPV-16 and 18 high-risk positive infection result; when the C line does not show color, it is determined that the test strip is invalid;

[0065] Figure 13 Specific detection result for Escherichia coli;

[0066] Figure 14 Specific detection result for Staphylococcus aureus;

[0067] Figure 15 Specific detection result for Pseudomonas aeruginosa;

[0068] Figure 16 Specific detection result for Candida albicans;

[0069] Figure 17 Sensitivity detection result for HPV high risk type;

[0070] Figure 18 Sensitivity detection result for HPV-16;

[0071] Figure 19 Sensitivity detection result for HPV-18;

[0072] Figure 20 Fluorescent PCR result for HPV high risk type;

[0073] Figure 21 Fluorescent PCR result for HPV-16;

[0074] Figure 22 Fluorescent PCR result for HPV-18;

[0075] Figure 23 High value repeatability detection result for HPV high risk type;

[0076] Figure 24 Medium value repeatability detection result for HPV high risk type;

[0077] Figure 25 Low value repeatability detection result for HPV high risk type;

[0078] Figure 26 High value repeatability detection result for HPV-16;

[0079] Figure 27 Medium value repeatability detection result for HPV-16;

[0080] Figure 28 Low value repeatability detection result for HPV-16;

[0081] Figure 29 High value repeatability detection result for HPV-18;

[0082] Figure 30 Medium value repeatability detection result for HPV-18;

[0083] Figure 31 HPV-18 low value repetitive detection results.

[0084] Reference signs:

[0085] 1 is a box body, 101 is an upper box body, 102 is a lower box body, 2 is a constant temperature heating element, 3 is a first stepped tube, 301 is a first sliding block, 302 is a second sliding rail, 303 is a second limiting boss, 4 is a second stepped tube, 401 is a second sliding block, 402 is a sealing boss, 5 is an end cover, 501 is a dropper, 6 is a sealing plug, 7 is a colloidal gold test paper, 8 is an observation window, 9 is a liquid outlet, 10 is a tube seat, 1001 is a first sliding rail, 1002 is a first limiting boss, 11 is a reaction bin, 12 is a TYPE-C interface, and 13 is a colloidal gold test paper placement table. DETAILED DESCRIPTION

[0086] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments, and they should not be understood as limiting the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. In the description of the present application, it should be understood that the terms used are only for the purpose of description, and should not be understood as indicating or implying relative importance.

[0087] In the following examples, the experimental methods are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0088] Example 1, preparation of a portable detection device based on constant temperature PCR reaction

[0089] Please refer to Figures 1 to 4 The portable detection device based on constant temperature PCR reaction provided in the embodiment comprises a box body 1, a constant temperature heating element 2, a first stepped tube 3, a second stepped tube 4, an end cover 5 and a sealing plug 6.

[0090] The box body 1 is internally provided with a colloidal gold test paper 7. The box body 1 is provided with an observation window 8, which is located above the colloidal gold test paper 7. The box body 1 is provided with a liquid outlet 9 at the top. The upper surface of the box body 1 is provided with a tube seat 10. The liquid outlet 9 is located in the tube seat 10.

[0091] The constant temperature heating element 2 is arranged on the top surface inside the box body 1 and located below the tube seat 10.

[0092] The lower part of the first stepped tube 3 is connected with the tube base 10, and the bottom surface of the first stepped tube 3 is provided with a reaction chamber 11 penetrating upward and downward;

[0093] The lower part of the second stepped tube 4 is connected with the first stepped tube 3, and the bottom surface of the second stepped tube 4 is provided with a liquid inlet;

[0094] The end cover 5 is buckled on the top of the second stepped tube 4, and the end cover 5 is provided with a liquid drop pipe 501, and the bottom end of the liquid drop pipe 501 is located in the liquid inlet;

[0095] The sealing plug 6 is plugged in the liquid drop port of the liquid drop pipe 501.

[0096] Specifically, the box body 1, the first stepped tube 3, the second stepped tube 4 and the tube base 10 in the whole shell of the detection device are all made of thermoplastic plastic ABS, which has high strength and good mechanical properties and thermal properties, and can meet the requirement that the shell does not deform at a temperature of 70℃ in the process of constant temperature PCR reaction. The ABS has good heat conduction performance, which can make the enzyme fully react in the reaction chamber 11.

[0097] In this embodiment, the box body 1 is a square box body, and one side of the box body 1 is provided with a TYPE-C interface 12 connected with the constant temperature heating element 2.

[0098] Specifically, the constant temperature heating element 2 adopts a ceramic resistance heating sheet in the existing technology product, for example, an electric heating wire core constant temperature heating sheet, which can realize constant output of 70℃ at 5V working voltage. Through the heat conduction characteristics of the ABS material, the temperature of the reaction chamber 11 is above 60 degrees, but will not exceed 70 degrees, which ensures the normal occurrence of enzyme reaction. The constant temperature heating element 2 is powered through the TYPE-C interface 12, and the TYPE-C interface 12 can be powered by a power bank or a mobile phone charger, and the heating time is 15-20 minutes.

[0099] Please refer to Figures 5 to 6 In this embodiment, the box body 1 includes an upper box body 101 and a lower box body 102 connected with each other.

[0100] Specifically, the upper box body 101 and the lower box body 102 are connected by a socket buckle or by a screw connection, and the observation window 8 is provided on the upper box body 101, and a transparent plastic film is arranged on the observation window 8, facilitating observation of the display result of the colloidal gold test paper 7, and simultaneously playing a sealing role. The colloidal gold test paper 7 is prearranged in the box body 1, and the bottom of the lower box body 102 is provided with a colloidal gold test paper placement table 13, the colloidal gold test paper placement table 13 is provided with a clamping groove, and the colloidal gold test paper 7 is placed in the clamping groove of the colloidal gold test paper placement table 13. The colloidal gold test paper placement table 13 can not only support the colloidal gold test paper 7, but also can limit the colloidal gold test paper 7 from moving in the box body 1, thereby affecting the observation of the measurement result.

[0101] In this embodiment, the side wall of the pipe seat 10 is provided with a first sliding rail 1001, and the first stepped pipe cylinder 3 is provided with a cylindrical first sliding block 301 which can slide in the first sliding rail.

[0102] Specifically, the pipe seat 10 and the upper box body 101 are integrally formed, and the pipe seat 10 is a circular pipe, the bottom surface of which is the upper surface of the upper box body 101. The liquid outlet 9 is arranged at a position other than the center of the bottom surface of the pipe seat 10, and can be a square opening. The top edge of the liquid outlet 9 is provided with a chamfer, facilitating the flow of liquid into the liquid outlet 9. The bottom of the liquid outlet 9 extends into the box body 1, so that the liquid can flow onto the colloidal gold test paper 7 through the liquid outlet 9, and the colloidal gold test paper 7 is located below the liquid outlet 9.

[0103] Two symmetrical first sliding rails 1001 are arranged on the side wall of the pipe seat 10, and first limiting bosses 1002 are arranged at the two ends of the first sliding rails 1001 respectively. The first limiting bosses 1002 and the end faces of the first sliding rails 1001 form a limiting groove. When the first sliding block 301 is clamped in the limiting groove, the position of the first sliding block 301 in the first sliding rail 1001 can be limited. When the first sliding rail 1001 is located in the limiting groove at one end, the reaction chamber 11 is aligned with the liquid outlet 9. When the first sliding rail 1001 is located in the limiting groove at the other end, the bottom surface of the reaction chamber 11 is in sealing contact with the bottom surface of the pipe seat 10, so that the liquid in the reaction chamber 11 cannot flow out from the bottom end thereof. Moreover, the reaction chamber 11 is located directly above the constant-temperature heating element 2, so that the reaction chamber 11 can be heated better.

[0104] The first stepped pipe cylinder 3 is a stepped cylinder, and two first sliding blocks 301 are symmetrically arranged in the respective first sliding rails 1001. Rotating the first stepped pipe cylinder 3 can switch the two limiting grooves of the first sliding block 301 in the first sliding rail 1001.

[0105] Please refer to Figures 7 to 8 In this embodiment, the inner wall of the reaction chamber 11 is provided in the form of a rack.

[0106] Specifically, the reaction bin 11 is arranged in the first stepped tube 3, and the upper end and the lower end of the reaction bin 11 are both open. The inner wall of the reaction bin 11 is uniformly arranged in a rack shape, which is similar to an internal gear structure. The reaction bin 11 is pre-added with amplification enzyme freeze-dried microspheres. The rack-shaped structure prevents the amplification enzyme freeze-dried microspheres from moving, so that the sample dropped in does not contact the microspheres. The top end and the bottom end of the reaction bin 11 are both provided with an annular clamping groove, and a sealing ring is arranged in the clamping groove. The sealing ring can keep the bottom surface of the tube seat 10 and the bottom surface of the second stepped tube 4 sealed.

[0107] Please refer to Figures 9 to 10 In this embodiment, the side wall of the first stepped tube 3 is provided with a second sliding rail 302, and the second stepped tube 4 is provided with a cylindrical second sliding block 401, which can slide in the second sliding rail 302. The two ends of the second sliding rail 302 are respectively provided with a second limiting boss 303, which is used to limit the position of the second sliding block 401 in the second sliding rail 302.

[0108] Specifically, the side wall of the first stepped tube 3 is provided with two symmetrically arranged second sliding rails 302, and the two ends of the second sliding rail 302 are respectively provided with a second limiting boss 303. The end face of the second limiting boss 303 and the end face of the second sliding rail 302 form a limiting groove. When the second sliding block 401 is clamped in the limiting groove, the position of the second sliding block 401 in the second sliding rail 302 can be limited. When the second sliding block 401 is located in the limiting groove at one end, the reaction bin 11 is just aligned with the liquid drop pipe 501. The bottom surface of the second stepped tube 4 is provided with a sealing boss 402, which is used to cover the top end of the reaction bin after the second stepped tube 4 is rotated. When the second sliding block 401 is located in the limiting groove at the other end, the top surface of the reaction bin 11 contacts the sealing boss 402 of the second stepped tube 4, so as to ensure the sealing property between the reaction bin 11 and the bottom surface of the second stepped tube 4.

[0109] The second stepped tube 4 is also a stepped cylinder, and the end cover 5 is buckled on the top of the second stepped tube 4. The space formed by the end cover 5 and the second stepped tube 4 is a buffer area, which can prevent the pathogen aerosol from diffusing to the external environment. The liquid drop pipe 501 of the end cover 5 is tubular, and the bottom end of the liquid drop pipe 501 is directly inserted into the liquid inlet.

[0110] In this embodiment, the sealing plug 6 is a rubber plug, and the sealing plug 6 is provided with a connecting head. The end cover 5 is provided with a connecting hole, and the connecting head is clamped in the connecting hole, so as to avoid the loss of the sealing plug 6 after the sealing plug 6 is taken off from the liquid drop pipe 501.

[0111] When the portable detection device based on the constant temperature PCR reaction of the embodiment is used to detect HPV virus, the detection device is a disposable device, the amplification enzyme freeze-dried microspheres are pre-placed in the reaction bin 11, the bottom end of the preset reaction bin 11 is in sealed contact with the tube seat 10, and the top end of the reaction bin 11 is aligned with the dropper 501; the sealing plug 6 is removed, two to three drops of about 40 microliters of sample are taken, are dropped into the dropper 501, and after 10 seconds, the sealing plug 6 is buckled again, at this time, the sample has entered the reaction bin 11, and the amplification enzyme freeze-dried microspheres are dissolved in the reaction bin 11. The space of the reaction bin 11 is about 0.5 ml, when the sample is mixed with the enzyme, the second stepped tube 4 is rotated to seal the top end of the reaction bin 11, at this time, the reaction bin 11 is a closed bin. The constant temperature heating element 2 is powered on to heat the reaction bin 11, and the heating time is 15 to 20 minutes to ensure the normal occurrence of the enzyme reaction. The second stepped tube 4 is rotated to align the dropper 501 with the reaction bin 11, the sealing plug 6 is removed, the diluent is injected into the reaction bin 11, the diluent is about 50 times the sample, the diluent is about 2 milliliters, after the diluent is injected, the sealing plug 6 is buckled again to prevent the aerosol containing pathogens from diffusing out of the shell; at the same time, the first stepped tube 3 is rotated to align the bottom end of the reaction bin 11 with the liquid outlet 9, the diluted sample is absorbed by the colloidal gold test paper 7 through the liquid outlet 9, the detection is completed through the water absorption layer of the colloidal gold test paper 7, and the detection result is displayed. The detection personnel observe the detection result through the observation window 8.

[0112] The preparation method of the colloidal gold test paper 7 for HPV nucleic acid detection comprises the following steps:

[0113] (1) Preparation of colloidal gold (AuNPs) and gold-labeled capture probes:

[0114] AuNPs are prepared by the trisodium citrate reduction method, and the particle size is 40 nm. 2.0 mL of a sodium citrate solution with a concentration of 194 mmol / L is quickly added to 100 mL of a boiling 0.1% HAuCl4 solution. When the color of the solution changes from purple to red, it indicates the formation of AuNPs. The solution is cooled to room temperature and stored at 4°C.

[0115] Preparation of gold-labeled capture probe: 100 μmol / L of anti-FAM monoclonal antibody 20 μL was slowly added to 500 μL of AuNPs with a size of 40 nm, and incubated in the dark for 16 h, then 10 mmol / L of phosphate buffer 56 μL (PB Buffer) (NaH2PO4 / Na2HPO4, pH=7.4) and 2 mol of NaCl solution 92 uL were added, and incubated for another 8 h, then centrifuged for 30 min (4 ℃, 16100 g) to remove the supernatant, and the precipitate was washed with 0.3 mol / L NaCl solution and 10 mmol / L PB Buffer. Finally, the precipitate was dissolved in 10 mmol / L PB Buffer containing 0.3 mol / L NaCl, and stored at 4 ℃ in the dark.

[0116] (2) Preparation of nitrocellulose membrane (NC membrane): 1.0 mg / mL of goat anti-mouse IGg polyclonal antibody was sprayed on the NC membrane to form a quality control line C; 0.8 mg / mL of anti-FITC monoclonal antibody was sprayed on the NC membrane to form a detection line T1, 0.8 mg / mL of anti-biotin monoclonal antibody was sprayed on the NC membrane to form a detection line T2, and 0.8 mg / mL of anti-digoxin monoclonal antibody was sprayed on the NC membrane to form a detection line T3.

[0117] (3) The water absorption paper, the treated sample pad, and the obtained gold-labeled pad, NC membrane were placed in a certain order on a PVC base plate, and the assembled PVC base plate was placed in a 37 ℃ oven for drying for 12 h, and then cut into strips for assembly to form the colloidal gold test paper 7 for HPV nucleic acid detection.

[0118] Example 2, primer composition for detecting HPV designed based on isothermal amplification reaction

[0119] 1. The L1 gene conservative region gene sequences in the genome sequences of HPV16 type (GenBank number NC_001526.4) and HPV18 type (GenBank number AY262282.1) were selected, and the selected conservative region sequences are shown in the following table:

[0120]

[0121] 2. Based on the above selected target sequences, the primer composition shown in Table 2 was designed.

[0122]

[0123] In Table 2, R represents A+G; Y represents C+T; M represents A+C; K represents G+T; S represents C+G; W represents A+T; H represents A+C+T; B represents C+G+T; V represents A+C+G; D represents A+G+T; N represents A+C+G+T. The underlined indicates n(a) or n(c). HPV-P and HPV-D are used to detect HPV high-risk types, and the HPV high-risk types include at least one of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68.

[0124] The 5' end of the upstream primers are all labeled with FAM to facilitate tracking and detection of the presence of HPV genes in experiments; the 5' end of the HPV16 downstream primer is labeled with biotin (Biotin), the 5' end of the HPV-18 downstream primer is labeled with digoxin (Digoxigenin), and the 5' end of the HPV high-risk type downstream primer is labeled with fluorescein isothiocyanate (FITC), which facilitates the capture or detection of HPV-specific probes during the detection process.

[0125] 3. HPV virus detection based on the above primer composition

[0126] The DNA constant temperature rapid amplification kit is used for amplification in a constant temperature amplification device. Each 25 μL reaction mixture includes 2xLAMP Premix Buffer (purchased from Baorui Biology, item number HW205-P01) 12.5 μL, 10xPrimer mix 2.5 μL (including 10 μM HPV16-P, 10 μM HPV16-D, 10 μM HPV18-P, 22 μM HPV18-D, 10 μM HPV-P, 10 μM HPV-D in TE Buffer), Bst 2.0 HS (8U / μL) 1 μL, RNase H II (50mU / μL) 0.25 μL, ddH2O 3.75 μL. After mixing the reaction solution, small batches are produced and stored in the form of freeze-dried balls (such as Figure 11 ).

[0127] When detecting HPV, 5 μL of positive quality control is added and mixed, and then added to the constant temperature amplification device, and incubated at 60°C for 20 min; after the reaction is completed, 1 mL of sample diluent is added to the device, the device is rotated, the reaction product is contacted with the sample end of the colloidal gold test strip, and the results are observed after 15 min. The detection line and the corresponding judgment results are shown in Figure 12 .

[0128] Example 3, specific detection

[0129] Common pathogens sharing the same infection site as HPV, such as Escherichia coli (CMCC44102), Staphylococcus aureus (CMCC26003), Pseudomonas aeruginosa (CMCC10104), and Candida albicans (CMCC98001), were selected as specific quality control samples. Inactivated standard control bacteria and inactivated clinical positive samples were used. The standard bacteria were purchased from the China Medical Bacteriological Collection Center (CMCC). Specificity was tested using this method, with enzyme-free sterile water as a blank control. Each sample was tested in triplicate. The specificity of the established detection method was determined based on the test strip results.

[0130] The results are as follows Figures 13-16 As shown, the test results for all four samples were negative, indicating that the method provided by this invention is specific.

[0131] Example 4: Sensitivity Detection

[0132] HPV-16 gDNA quality control material (product number: GBW(E)091308, content: 4×10) purchased from Jingliang Technology (Shenzhen) Co., Ltd. was used. 7 copies / mL), HPV-18 gDNA quality control (No.: GBW(E)091309, content: 4×10⁻⁶). 7 (copies / mL), HPV high-risk positive plasmid quality control (content 5×10) 7 To perform sensitivity testing, 1 mL of RNase-free water was reconstituted, and then serially diluted 10-fold with RNase-free water to obtain concentrations of 10... 7 -10 2 DNA solutions of different concentrations (copies / mL) were used as templates, and the detection was performed using the method provided in Example 2. The detection results are as follows: Figures 17-19 As shown.

[0133] Meanwhile, the above samples were tested using a standard fluorescent PCR kit (15 high-risk human papillomavirus nucleic acid detection kit (PCR-fluorescent probe method), purchased from Sansure Biotech, National Medical Device Registration Certificate 20163401304). The test results are as follows: Figures 20-22 As shown.

[0134] according to Figures 17-22 It can be seen that the method works when the target nucleic acid concentration is 10. 2 It can still stably generate amplification signals even at copies / mL, and its detection sensitivity is at the same level as existing fluorescent PCR methods (such as real-time fluorescent PCR), and it has equivalent detection capabilities.

[0135] Example 5: Repeatability Test

[0136] HPV-16 gDNA quality control material (product number: GBW(E)091308, content: 4×10) purchased from Jingliang Technology (Shenzhen) Co., Ltd. was used. 7 copies / mL), HPV-18 gDNA quality control (No.: GBW(E)091309, content: 4×10⁻⁶). 7 copies / mL), HPV high-risk plasmid quality control sample (content 5×10⁻⁶). 7 For repeatability testing, after reconstitution with 1 mL of RNase-free water, the solution was serially diluted 10-fold with nucleic acid-negative human defibrinated serum to obtain high (1×10) copies / mL. 6 copies / mL), medium (1×10) 4 copies / mL), low (1×10) 2 Three concentrations of standards (copies / mL) were tested three times in each round of testing, and the results were observed to be consistent.

[0137] The results are as follows Figures 23-31 As shown, the test results of the standard samples at high, medium, and low concentrations are consistent, indicating that the method provided by this invention has good consistency.

[0138] In the description of this invention, it should be noted that the terms "upper", "lower", "front", "rear", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the system or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0139] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "assembly," "setup," and "connection" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0140] In the description of the present application, it is necessary to note that the relationship terms such as first or second and the like are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require the existence of such actual relationship or order between these entities or operations. Moreover, the terms "comprise", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes elements not explicitly listed, and also includes elements inherent in such process, method, article or equipment. Without more limitations, the element defined by the statement "comprising a" does not exclude the existence of other same elements in the process, method, article or equipment including the element.

[0141] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A primer composition for detecting human papillomavirus, characterized by, The primer composition comprises a first primer, a second primer, a third primer, a fourth primer, a fifth primer and a sixth primer, wherein: the first primer is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO: 15 modified with a fluorescent group FAM at the 5' end; the second primer is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO: 16 modified with biotin at the 5' end; the third primer is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO: 17 modified with a fluorescent group FAM at the 5' end; the fourth primer is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO: 18 modified with digoxin at the 5' end; the fifth primer is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO: 19 modified with a fluorescent group FAM at the 5' end; the sixth primer is a single-stranded DNA molecule with a nucleotide sequence shown as SEQ ID NO: 20 modified with a fluorescent group FITC at the 5' end.

2. A product characterized by, The primer composition of claim 1.

3. Use of the primer composition of claim 1 or the product of claim 2 in the preparation of a product for detecting human papillomavirus.

4. A method for detecting the presence or absence of human papillomavirus in a test sample for non-disease diagnostic purposes, characterized by, The method comprises: extracting DNA of a sample to be detected to obtain a DNA sample to be detected; performing an amplification reaction on the DNA sample to be detected using the primer composition of claim 1 to obtain an amplification product; detecting the amplification product to determine whether the sample to be detected contains human papillomavirus according to the detection result.

5. The method of claim 4, wherein, The human papillomavirus comprises at least one of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68.

6. The method of claim 4, wherein, The amplification reaction is performed in a constant temperature environment of 55-65℃.

7. The method of claim 4, wherein, The amplification product is added dropwise to a sample area of a colloidal gold immunochromatography test strip, and whether the sample to be detected contains human papillomavirus is determined by whether the corresponding bands develop color.

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