CN113584229B - Primer probe combination, kit and application for isothermal nucleic acid amplification detection of human papilloma virus type 6 and/or type 11 - Google Patents
Primer probe combination, kit and application for isothermal nucleic acid amplification detection of human papilloma virus type 6 and/or type 11 Download PDFInfo
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Abstract
The invention provides a primer probe combination, a kit and application for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11, and belongs to the technical field of molecular biology. The invention provides a primer probe combination for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11, which comprises a first primer probe group and/or a second primer probe group; the first primer probe group comprises a specific primer pair aiming at HPV6 and a target gene probe aiming at HPV 6; the second primer probe set comprises a specific primer pair for HPV11 and a target gene probe for HPV11. The kit prepared by the primer probe composition provided by the invention can be used for rapidly detecting HPV6 and/or HPV11 viruses at normal temperature, has high sensitivity and good specificity, is easy to popularize and apply in a large range, and has wide market prospect and great economic and social benefits.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer probe combination, a kit and application for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11.
Background
Condyloma acuminatum (condyloma acuminate, CA) is squamous epithelial verrucous hyperplasia of external genitalia, anus and other parts caused by specific HPV infection, is common in adult population in sexual activity period, has high global incidence rate, is second to epidemic disease, and has an increasing trend in recent years, and human papillomavirus (humanpapilloma virus, HPV) infection is a main factor causing disease. HPV is a circular DNA virus belonging to the papillomavirus family that can cause benign and malignant tumors in human mucosal tissues. The HPV types found at present are more than 150. The major viral subtypes responsible for condyloma acuminatum infection are HPV6 and HPV11. Therefore, the rapid and accurate detection of HPV6 and HPV11 type is of great significance for early diagnosis, early treatment and prevention of condyloma acuminatum.
Human papillomaviruses cannot be cultured in vitro, and the current laboratory diagnosis methods of HPV mainly comprise cytological examination, molecular biological examination, colposcopy examination, pathological examination and the like. Cytological examination is a gold standard for diagnosis of HPV infection. However, conventional cytological examination makes it difficult to distinguish virus infection at an early stage, so that the probability of false negatives is high. Furthermore, the range of observation of papanicolaou staining is limited, reaching the peak of sensitivity. Colposcopy and pathology tests are less specific to morphologically atypical individuals and are subject to interference from quality of the slide and subjective factors of the reader. Various molecular detection techniques for HPV screening have been developed, such as polymerase chain reaction (Polymerase chain reaction, PCR), immunochromatographic assay (immunochromatographic), in situ hybridization (In situ hybridization, ISH), and the like. The second generation hybrid capture technology is widely used for HPV detection, has high specificity and sensitivity to the screening of condyloma acuminatum, but has the biggest defects of being incapable of specific typing and generating cross reaction. PCR is only suitable for specific facilities and laboratories because it requires accurate temperature control equipment and complex analytical instrumentation, as well as a high level of expertise, and the special equipment and cumbersome procedures required remain a problem for field detection, limiting widespread adoption at the base level. Since the existing methods for detecting HPV6 and HPV11 viruses are insufficient to meet the requirements for rapid detection of HPV6 and HPV11 viruses, it is necessary to develop a method capable of rapidly detecting HPV6 viruses at normal temperature for use in a basic layer.
Disclosure of Invention
In order to solve the problems, the invention provides a primer probe combination, a kit and application for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11. The primer probe combination for isothermal nucleic acid amplification detection of human papilloma virus type 6 and/or type 11 can realize rapid detection of HPV6 and HPV11 viruses, does not need special equipment, has simple operation process, and is very suitable for application in a basic layer.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a group of primer probe combinations for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11, which comprises a first primer probe group and/or a second primer probe group; the first primer probe group comprises a specific primer pair aiming at HPV6 and a target gene probe aiming at HPV 6; the second primer probe set comprises a specific primer pair for HPV11 and a target gene probe for HPV 11;
the specific primer pair for HPV6 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 2; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 3; the target gene probe for HPV6 comprises a nucleotide sequence shown in SEQ ID No. 1;
the specific primer pair for HPV11 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 10; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 11; the target gene probe for HPV11 comprises a nucleotide sequence shown in SEQ ID No. 9.
Preferably, the target gene of the specific primer pair for HPV6 comprises a nucleotide sequence as shown in SEQ ID No. 8; the target gene of the specific primer pair for HPV11 comprises a nucleotide sequence shown as SEQ ID No. 16.
Preferably, the kit further comprises an endogenous internal reference primer probe set; the endogenous reference primer probe group comprises an endogenous reference probe and an endogenous reference primer pair; the endogenous reference probe comprises a nucleotide sequence shown as SEQ ID No. 17; the forward primer of the endogenous internal reference primer pair comprises a nucleotide sequence shown as SEQ ID No. 18; the reverse primer of the endogenous reference primer pair comprises a nucleotide sequence shown as SEQ ID No. 19.
Preferably, the target gene of the endogenous reference primer pair comprises a nucleotide sequence as shown in SEQ ID No. 20.
Preferably, the type of endogenous reference probe comprises an exo probe.
The invention provides application of the primer probe combination in the technical scheme in preparing a tool for detecting human papillomavirus type 6 and/or type 11.
The invention provides a kit for detecting human papillomavirus type 6 and/or type 11, which comprises the primer probe combination in the technical scheme.
Preferably, the target gene probe for HPV6, the target gene probe for HPV11 and the endogenous reference probe are labeled with fluorescent groups having different fluorescence, respectively; the fluorescent group includes FAM, HEX, ROX, TET, JOE, CY, CY5, TAMRA or VIC.
Preferably, the isothermal amplification temperature of the kit is 39-42 ℃; the isothermal amplification time of the kit is 15-30 min.
The invention provides application of the kit in detecting human papillomavirus type 6 and/or type 11 in non-diagnostic purpose, when detecting HPV6, the first primer probe set and the endogenous internal reference primer probe set are combined; when detecting HPV11, combining the second primer probe group with the endogenous internal reference primer probe group; when HPV6 and HPV11 are detected simultaneously, the first primer probe set and the second primer probe set are combined, and meanwhile, an endogenous internal reference primer probe set is independently provided with a tube to serve as an internal reference.
The beneficial effects are that:
the invention provides a group of primer probe combinations for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11, which comprises a first primer probe group and/or a second primer probe group; the first primer probe group comprises a specific primer pair aiming at HPV6 and a target gene probe aiming at HPV 6; the second primer probe set comprises a specific primer pair for HPV11 and a target gene probe for HPV 11; the specific primer pair for HPV6 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 2; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 3; the target gene probe for HPV6 comprises a nucleotide sequence shown in SEQ ID No. 1; the specific primer pair for HPV11 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 10; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 11; the target gene probe for HPV11 comprises a nucleotide sequence shown in SEQ ID No. 9. The kit prepared by the primer probe composition provided by the invention can be used for rapidly detecting HPV6 and/or HPV11 viruses at normal temperature, has high sensitivity and good specificity, is easy to popularize and apply in a large range, and has wide market prospect and great economic and social benefits.
Drawings
FIG. 1 shows the detection results of HPV6 different primer probe combinations, wherein 1 is the amplified fluorescent signal of the SEQ ID No.1,2,3 combination; 2 is the amplified fluorescent signal of the combination of SEQ ID No.1,2, 6; 3 is the amplified fluorescent signal of the combination of SEQ ID No.1,2 and 7; 4 is the amplified fluorescent signal of the combination of SEQ ID No.1,4, 3; 5 is the amplified fluorescent signal of the combination of SEQ ID No.1,4, 6; 6 is the amplified fluorescent signal of the combination of SEQ ID No.1,4, 7; 7 is the amplified fluorescent signal of the combination of SEQ ID No.1,5, 3; 8 is the amplified fluorescent signal of the combination of SEQ ID No.1,5 and 6; 9 is the amplified fluorescent signal of the combination of SEQ ID No.1,5, 7; negative: a negative control;
FIG. 2 shows the detection results of HPV11 different primer probe combinations, wherein 1 is the amplified fluorescent signal of the SEQ ID No.9, 10, 11 combination; 2 is the amplified fluorescent signal of the combination of SEQ ID No.9, 10, 14; 3 is the amplified fluorescent signal of the combination of SEQ ID No.9, 10, 15; 4 is the amplified fluorescent signal of the combination of SEQ ID No.9, 12, 11; 5 is the amplified fluorescent signal of the combination of SEQ ID No.9, 12, 14; 6 is the amplified fluorescent signal of the combination of SEQ ID No.9, 12, 15; 7 is the amplified fluorescent signal of the combination of SEQ ID No.9, 13, 11; 8 is the amplified fluorescent signal of the combination of SEQ ID No.9, 13, 14; 9 is the amplified fluorescent signal of the combination of SEQ ID No.9, 13, 15; negative: a negative control;
FIG. 3A is a graph of HPV6 positive amplification fluorescence signals in example 2;
FIG. 3B is a graph showing fluorescence signals of amplification of reference DNA in example 2;
FIG. 4A is a graph of HPV11 positive amplification fluorescence signal in example 2;
FIG. 4B is a graph showing fluorescence signals of amplification of reference DNA in example 2;
FIG. 5A is a graph of HPV6 positive amplified fluorescence signals from example 3;
FIG. 5B is a graph showing fluorescence signals of amplification of reference DNA in example 3;
FIG. 6A is a graph of HPV11 positive amplified fluorescence signals from example 3;
FIG. 6B is a graph of fluorescence signal of internal DNA amplification in example 3;
FIG. 7A is a graph showing positive amplified fluorescent signals of HPV6 at different concentrations at an internal reference concentration of 100 copies/. Mu.L;
FIG. 7B is a graph showing amplified fluorescence signals of reference DNA at a reference concentration of 100 copies/. Mu.L;
FIG. 8A shows an internal reference concentration of 10 5 Amplification fluorescent signal patterns of HPV6 at different concentrations at copy/. Mu.L;
FIG. 8B shows an internal reference concentration of 10 5 Amplification fluorescent signal plot of reference DNA at copy/μl;
FIG. 9A is a graph showing positive amplified fluorescent signals of HPV11 at different concentrations at an internal reference concentration of 100 copies/. Mu.L;
FIG. 9B is a graph showing amplified fluorescence signals of reference DNA at a reference concentration of 100 copies/. Mu.L;
FIG. 10A shows an internal reference concentration of 10 5 Amplification fluorescence signal plots of HPV11 at different concentrations at copy/. Mu.L;
FIG. 10B shows an internal reference concentration of 10 5 Amplification fluorescent signal plot of reference DNA at copy/μl;
FIG. 11A is a graph of amplified fluorescent signals of HPV6 and other human papillomaviruses in example 5, wherein 1 is an HPV6 amplification curve; 2 is the amplification curve of other human papillomaviruses;
FIG. 11B is a graph showing the amplified fluorescent signals of internal reference DNA corresponding to HPV6 groups of different viruses in example 5;
FIG. 12A is a graph of amplified fluorescent signals of HPV11 and other human papillomaviruses of example 5, wherein 1 is an HPV6 amplification curve; 2 is the amplification curve of other human papillomaviruses;
FIG. 12B is a graph showing the amplified fluorescent signals of internal DNA corresponding to HPV11 group of different viruses according to example 5.
Detailed Description
The invention provides a group of primer probe combinations for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11, which comprises a first primer probe group and/or a second primer probe group; the first primer probe group comprises a specific primer pair aiming at HPV6 and a target gene probe aiming at HPV 6; the second primer probe set comprises a specific primer pair for HPV11 and a target gene probe for HPV 11;
the specific primer pair for HPV6 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 2; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 3; the target gene probe for HPV6 comprises a nucleotide sequence shown in SEQ ID No. 1;
the specific primer pair for HPV11 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 10; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 11; the target gene probe for HPV11 comprises a nucleotide sequence shown in SEQ ID No. 9.
The primer probe combination for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11 is designed according to a conserved region with high homology of HPV6 and HPV11, and a target sequence suitable for detecting HPV6 and HPV11 viruses is determined. In the present invention, the target gene of the specific primer pair for HPV6 includes the nucleotide sequence shown as SEQ ID No. 8. In the present invention, the target gene of the specific primer pair for HPV11 comprises the nucleotide sequence shown in SEQ ID No. 16.
The specific primer pair for HPV6 comprises a forward primer and a reverse primer. In the invention, the forward primer comprises a nucleotide sequence shown as SEQ ID No.2, and the specific nucleotide sequence is as follows: 5'-GAACTTATTACCAGTGTTATACAGGATGGC-3'. In the present invention, the reverse primer comprises a nucleotide sequence shown as SEQ ID No.3, and the specific nucleotide sequence is as follows: 5'-TGGGTCTGCAGCCATTTGTAAATAATCTGGA-3'. In the invention, the target gene probe for HPV6 comprises a nucleotide sequence shown in SEQ ID No.1, and the specific nucleotide sequence is as follows: 5'-ATGGTTGACACAGGCTTTGGTGCTATGAATTTTGCTGATTTGCAGAC-3'. The specific primer probe combination for HPV6 can accurately and efficiently detect HPV6 virus, has high sensitivity and good specificity, can realize amplification under the constant temperature condition, and is simple to operate and free of precise instruments.
The specific primer pair for HPV11 comprises a forward primer and a reverse primer. In the present invention, the forward primer comprises a nucleotide sequence shown as SEQ ID No.10, and the specific nucleotide sequence is as follows: 5'-AACCTTTAGGCGTTGGTGTTAGTGGGCATC-3'. In the present invention, the reverse primer comprises a nucleotide sequence shown as SEQ ID No.11, and the specific nucleotide sequence is as follows: 5'-TAATCCATACCTACATTA ACCCTATTATCC-3'. In the invention, the target gene probe for HPV11 comprises a nucleotide sequence shown in SEQ ID No.9, and the specific nucleotide sequence is as follows: 5'-TATGATGATGTAGAAAATAGTGGTGGGTATGGTGGTAATCCTGGTCA-3'. The specific primer probe combination for HPV11 can accurately and efficiently detect HPV11 virus, has high sensitivity and good specificity, can realize amplification under the constant temperature condition, and is simple to operate and free of precise instruments.
The primer probe combination for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or 11 also preferably comprises an endogenous internal reference primer probe group; the endogenous reference primer probe group comprises an endogenous reference probe and an endogenous reference primer pair. The invention designs an endogenous reference probe and a primer pair aiming at a housekeeping gene RNaseP, the gene is stable and almost exists in all specimens, no exogenous reference is added, a false negative result can be avoided, and the influence of the exogenous reference on a detection result is prevented. In the present invention, the endogenous reference probe preferably comprises a nucleotide sequence shown as SEQ ID No.17, specifically: 5'-ATGGTACACTTAAACTGGGGACTCTGGGGATGATGGCTCTTACTTCGT-3'. In the present invention, the forward primer of the endogenous reference primer pair preferably includes a nucleotide sequence shown as SEQ ID No.18, specifically: 5'-GCTTAAAATGTGTTCTAGCCTTGGCGTTCA-3'. In the present invention, the reverse primer of the endogenous reference primer pair preferably includes a nucleotide sequence shown in SEQ ID No.19, specifically: 5'-TCCAGAGGTTCAGTCTCTAAATTTTCCCCA-3'. The target gene of the endogenous reference primer pair of the present invention preferably includes a nucleotide sequence shown as SEQ ID No. 20. When detecting HPV6, the present invention preferably uses a combination of a first primer probe set and an endogenous reference primer probe set to create a dual isothermal amplification system containing endogenous reference. When detecting HPV11, the present invention preferably uses a second primer probe set in combination with an endogenous reference primer probe set to create a dual isothermal amplification system containing endogenous reference. When simultaneously detecting HPV6 and HPV11, the invention preferably combines the first primer probe set and the second primer probe set, and simultaneously, sets an endogenous internal reference primer probe set as an internal reference by independently arranging a tube so as to realize the function of the internal reference and avoid false positive results. In the present invention, the type of the endogenous reference probe preferably includes an exo probe.
According to the invention, through respectively carrying out gene sequencing and comparison on HPV6 and HPV11, respectively designing specific probes and primer pairs for detecting HPV6 and HPV11, designing endogenous internal reference probes and primer pairs for a human housekeeping gene RNaseP, establishing 2 double isothermal nucleic acid amplification systems containing endogenous internal references on the basis, and amplifying under isothermal conditions, the amplification of HPV6, HPV11 and internal reference DNA can be realized, and the sensitivity and the specificity are high; in addition, the addition of endogenous internal parameters effectively reduces the occurrence of false negative caused by reasons such as sample sampling, reagent kit failure or experimental operation, is more suitable for detection of a large number of samples, and is convenient for clinical application.
The invention provides application of the primer probe combination in the technical scheme in preparing a tool for detecting human papillomavirus type 6 and/or type 11. In the present invention, the means is preferably a kit.
The invention provides a kit for detecting human papillomavirus type 6 and/or type 11, which comprises the primer probe combination according to any one of the technical schemes. In the invention, the target gene probe for HPV6, the target gene probe for HPV11 and the endogenous reference probe are preferably labeled with fluorescent groups with different fluorescence, so as to realize accurate detection of HPV6 and HPV11. In the present invention, the fluorescent group preferably includes FAM, HEX, ROX, TET, JOE, CY, CY5, TAMRA or VIC. The method for labeling the fluorescent groups has no special requirement, and the method is only needed by adopting the conventional labeling method in the field.
When detecting HPV6, the present invention preferably uses a combination of a first primer probe set and an endogenous reference primer probe set to create a dual isothermal amplification system containing endogenous reference. When detecting HPV11, the present invention preferably uses a second primer probe set in combination with an endogenous reference primer probe set to create a dual isothermal amplification system containing endogenous reference. The amplification system of the kit of the present invention preferably comprises the following components in the concentration of 50. Mu.L for detection of HPV6 and HPV11, respectively: tris buffer 45mM; 80mM potassium acetate; magnesium acetate 14mM; dithiothreitol 4mM; polyethylene glycol 7.28% (w/v); ATP 5mM; dNTPs 0.24mM; creatine phosphate 30 μg/U; 500 ng/. Mu.L of single-chain binding protein; recombinant enzyme 400 ng/. Mu.L; uvsY protein 70 ng/. Mu.L; DNA polymerase 90 ng/. Mu.L; exonuclease 85 ng/. Mu.L; 120nM of target gene probe; target gene forward primer 420nM; target gene reverse primer 420nM; endogenous reference probe sequence 60nM; 200nM of endogenous reference gene forward primer; 200nM of endogenous reference gene reverse primer. In the present invention, the method of amplification of the kit is preferably isothermal amplification. In the present invention, the isothermal amplification temperature is preferably 39 to 42℃and more preferably 39 ℃. In the invention, the constant-temperature amplification time is preferably 15-30 min; further preferably 30min. The invention can realize amplification under the constant temperature condition, has short amplification time, does not need precise instruments, has lower requirements on technicians, is easy to operate, and is convenient for basic staff to detect and screen a large number of samples. The interpretation of the results of the present invention is preferably determined according to the following principle: when the detection results of the HPV6 target or HPV11 target and the internal reference DNA show amplified fluorescence signals, the detection result is positive; when the detection result of the HPV6 target or the HPV11 target does not generate an amplified fluorescent signal, the detection result of the internal reference DNA generates an amplified fluorescent signal, and the detection result is negative; when the detection result of the HPV6 target or the HPV11 target shows an amplified fluorescent signal, the detection result of the internal reference DNA shows no amplified fluorescent signal, and the detection result is positive; when no amplified fluorescent signal appears on the HPV6 target or HPV11 target and the reference DNA detection results, the detection results may be false negative, and repeated experiments or DNA re-extraction are recommended for detection.
When the invention detects HPV6 and HPV11 simultaneously, the invention preferably combines the first primer probe set and the second primer probe set, and simultaneously, sets an endogenous internal reference primer probe set as an internal reference by a single tube, thereby avoiding the generation of false negative results. In the simultaneous detection, the two probes in the first primer probe set and the second primer probe set are preferably set to different fluorescence, and detection is performed in 2 different channels. In the present invention, the amplification conditions for simultaneous detection are preferably the same as those for separate detection.
The kit for detecting human papilloma virus type 6 and/or type 11 provided by the invention has the advantages that the HPV6 target gene sensitivity can reach 10 copies/mu L, the internal reference gene sensitivity can reach 100 copies/mu L, and weak positive clinical specimens with CT values larger than 35 can be detected; HPV11 target gene sensitivity can reach 100 copies/. Mu.L, reference gene sensitivity can reach 100 copies/. Mu.L, and weak positive clinical specimens with CT values greater than 35 can be detected; has no cross reaction to other types of human papillomavirus detection, has good specificity, is easy to popularize and apply in a large scale, and has wide market prospect and great economic and social benefits.
The invention provides application of the kit in detecting human papillomavirus type 6 and/or type 11 in non-diagnostic purpose, when detecting HPV6, combining a specific primer pair and a target gene probe aiming at HPV6 with an endogenous internal reference primer pair and a probe; when detecting HPV11, combining a specific primer pair and a target gene probe for HPV11 with an endogenous internal reference primer pair and a probe; when HPV6 and HPV11 are detected simultaneously, a specific primer pair and a target gene probe for HPV6 are used in combination with a specific primer pair and a target gene probe for HPV11, and simultaneously, an endogenous internal reference primer pair and a probe are separately provided as internal references. The invention can realize the detection of HPV6 and HPV11 simultaneously, can realize the independent detection of HPV6 and HPV11 respectively, has flexible combination, is efficient and accurate, is very suitable for the detection of a large number of samples, and is convenient for clinical application.
For further explanation of the present invention, a set of primer probe combinations, kits and applications for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or type 11 provided herein are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Specific primer pair, target probe, internal reference probe and design and determination of internal reference primer pair for isothermal nucleic acid amplification detection of HPV6 and HPV11 viruses
And respectively downloading all HPV6 and HPV11 virus whole genome sequences, performing sequence comparison, searching a conserved region with high homology, and determining target sequences suitable for detecting HPV6 and HPV11 viruses, wherein the nucleotide sequence suitable for detecting the target sequences of HPV6 viruses is shown as SEQ ID NO.8, and the nucleotide sequence suitable for detecting the target sequences of HPV11 viruses is shown as SEQ ID NO. 16. Designing a plurality of specific primers and target probes in a conservation region. RAA primer design principle: firstly, the primer length, the RAA primer is longer than a typical PCR primer, and the general requirement is 30-35 bp; secondly, a primer sequence, wherein the 5' -end (3-5 bp) avoids the occurrence of repeated G, and is preferably C or T; the 3' -end (last 3 bases) preferably has G and C; the GC content is not more than 70% or less than 30%; the formation of secondary structure, primer dimer, etc. is avoided between the primers. RAA probe design principle: the RAA fluorescent probe (exo probe) mainly comprises four special parts, a blocker (usually C3-spacer) at the 3' -end, abasic nucleotide analogues (tetrahydrofuran [ THF ] residues, sometimes called dSpacer) and fluorescent groups (dT-fluorescent groups) and quenching groups (dT-quenching groups) located on both sides of THF, and the distance between the two groups is about 2-5 bp. The probe is typically 46 to 52 bases, at least 30 bases from the 5 'end to the THF site, and at least 15 bases from the THF site to the 3' end.
Example 1-1
The nucleotide sequence of the target gene probe for HPV6 is shown as SEQ ID No.1, and the specific nucleotide sequence is as follows: 5'-ATGGTTGACACAGGCTTTGGTGCTATGAATTTTGCTGATTTGCAGAC-3'. The specific primer pair for HPV6 comprises a forward primer and a reverse primer; wherein the nucleotide sequence of the forward primer is shown as SEQ ID No.2, and the specific nucleotide sequence is as follows: 5'-GAACTTATTACCAGTGTTATACAGGATGGC-3'; the nucleotide sequence of the reverse primer is shown as SEQ ID No.3, and the specific nucleotide sequence is as follows: 5'-TGGGTCTGCAGCCATTTGTAAATAATCTGGA-3'. The nucleotide sequence of the endogenous reference probe is shown as SEQ ID No.17, and the specific nucleotide sequence is as follows: 5'-ATGGTACACTTAAACTGGGGACTCTGGGGATGATGGCTCTTACTTCGT-3'. The nucleotide sequence of the forward primer of the endogenous reference is shown as SEQ ID No.18, and the specific nucleotide sequence is as follows: 5'-GCTTAAAATGTGTTCTAGCCTTGGCGTTCA-3'. The nucleotide sequence of the reverse primer of the endogenous reference is shown as SEQ ID No.19, and the specific nucleotide sequence is as follows: 5'-TCCAGAGGTTCAGTCTCTAAATTTTCCCCA-3'.
Test example 1-1
To demonstrate the advantages of the specific primer pair of HPV6 of the present invention, the following comparative primers are presented, with specific nucleotide sequences shown in SEQ ID No. 4-SEQ ID No.7. The specific nucleotide sequence is as follows:
5’-CATTGGGGTAAAGGTAAACAGTGTACTAAT-3’，(SEQ ID No.4)；
5’-CGAGCATTGGGGTAAAGGTAAACAGTGTACTA-3’，(SEQ ID No.5)；
5’-AATCTGGATATTTACATGTAGTGCCACATA-3’，(SEQ ID No.6)；
5’-GTCTGCAGCCATTTGTAAATAATCTGGATA-3’，(SEQ ID No.7)。
the following target gene probe primer combinations were compared:
a first group: a combination of SEQ ID No.1,2,3 (invention);
second group: a combination of SEQ ID No.1,2, 6;
third group: a combination of SEQ ID No.1,2, 7;
fourth group: a combination of SEQ ID No.1,4, 3;
fifth group: the combination of SEQ ID No.1,4, 6;
sixth group: the combination of SEQ ID No.1,4, 7;
seventh group: a combination of SEQ ID No.1,5, 3;
eighth group: a combination of SEQ ID No.1,5, 6;
ninth group: the combination of SEQ ID No.1,5, 7;
the sensitivity and specificity of the above combinations were evaluated by comparison with HPV6 virus positive clinical samples according to the time to peak and fluorescence intensity, and the detection results are shown in FIG. 1.
As can be seen from FIG. 1, under the same conditions, the primer probe combinations of SEQ ID Nos. 1,2 and 3 of the present invention have better positive threshold time and fluorescence signal values than other combinations, and the positive threshold time is early and the fluorescence intensity is high, so that the probe primer combinations of the present invention are optimal.
Examples 1 to 2
The nucleotide sequence of the target gene probe for HPV11 is shown in SEQ ID No.9, and the specific nucleotide sequence is as follows: 5'-TATGATGATGTAGAAAATAGTGGTGGGTATGGTGGTAATCCTGGTCA-3'. The specific primer pair of the invention for HPV11 comprises a forward primer and a reverse primer; wherein the nucleotide sequence of the forward primer is shown as SEQ ID No.10, and the specific nucleotide sequence is as follows: 5'-AACCTTTAGGCGTTGGTGTTAGTGGGCATC-3'; the nucleotide sequence of the reverse primer is shown as SEQ ID No.11, and the specific nucleotide sequence is as follows: 5'-TAATCCATACCTACATTAACCCTATTATCC-3'. The nucleotide sequence of the endogenous reference probe is shown as SEQ ID No.17, and the specific nucleotide sequence is as follows: 5'-ATGGTACACTTAAACTGGGGACTCTGGGGATGATGGCTCTTACTTCGT-3'. The nucleotide sequence of the forward primer of the endogenous reference is shown as SEQ ID No.18, and the specific nucleotide sequence is as follows: 5'-GCTTAAAATGTGTTCTAGCCTTGGCGTTCA-3'. The nucleotide sequence of the reverse primer of the endogenous reference is shown as SEQ ID No.19, and the specific nucleotide sequence is as follows: 5'-TCCAGAGGTTCAGTCTCTAAATTTTCCCCA-3'.
Test examples 1 to 2
To demonstrate the advantages of the specific primer pair of HPV11 of the present invention, the following comparative primers are given, with specific nucleotide sequences shown in SEQ ID No. 12-SEQ ID No.15. The specific nucleotide sequence is as follows:
5’-CGTTTAGTATGGGCGTGCACAGGGTTGGAG-3’，(SEQ ID No.12)；
5’-TGTTTGACCCCACTACACAGCGTTTAGTAT-3’，(SEQ ID No.13)；
5’-ACAGCCCACCATACATAGCTGGGTTTGTTT-3’，(SEQ ID No.14)；
5’-GTGTACCCTTACCCCAATGTTCACCTAACG-3’，(SEQ ID No.15)。
the following target gene probe primer combinations were screened:
a first group: the SEQ ID No.9, 10, 11 combinations (invention);
second group: the combination of SEQ ID Nos. 9, 10, 14;
third group: the combination of SEQ ID No.9, 10, 15;
fourth group: the combination of SEQ ID No.9, 12, 11;
fifth group: the combination of SEQ ID Nos. 9, 12, 14;
sixth group: the combination of SEQ ID No.9, 12, 15;
seventh group: the combination of SEQ ID No.9, 13, 11;
eighth group: the combination of SEQ ID No.9, 13, 14;
ninth group: SEQ ID No.9, 13, 15.
The sensitivity and specificity of the above combinations were evaluated by comparison with HPV11 virus positive clinical samples according to the time to peak and fluorescence intensity, and the detection results are shown in FIG. 2.
As can be seen from FIG. 2, under the same conditions, the primer probe combinations of SEQ ID Nos. 9, 10 and 11 of the present invention have better positive threshold time and fluorescence signal values than other combinations, and the positive threshold time is early and the fluorescence intensity is high, so that the primer probe combinations of the present invention are optimal.
Example 2 internal control-containing double isothermal nucleic acid amplification method for detecting HPV6 and HPV11 according to the present invention
1. Sample origin and DNA extraction of HPV6 and HPV11
The virus sample is a specimen containing HPV6 and HPV11 live viruses, which is collected from cervical exfoliated cell samples of different patients by Tangshan workers' hospitals, and the DNA extraction adopts a Tianlong extraction kit, and the DNA extraction equipment is a Tianlong full-automatic nucleic acid extractor.
2. Primers and probes for detecting HPV6 virus (SEQ ID Nos. 1 to 3, SEQ ID Nos. 17 to 19) and primers and probes for detecting HPV11 virus (SEQ ID Nos. 9 to 11, SEQ ID Nos. 17 to 19) determined in example 1, which are suitable for the isothermal nucleic acid amplification method, were used, wherein the target probe was labeled with FAM fluorophore and the internal reference probe was labeled with HEX fluorophore.
3. Preparing an amplification system: the isothermal nucleic acid amplification system was prepared in a 200. Mu.L centrifuge tube (50. Mu.L in volume) according to the following proportions:
tris buffer 45mM; 80mM potassium acetate; magnesium acetate 14mM; dithiothreitol 4mM; polyethylene glycol 7.28% (w/v); ATP 5mM; dNTPs 0.24mM; creatine phosphate 30 μg/U; 500 ng/. Mu.L of single-chain binding protein; recombinant enzyme 400 ng/. Mu.L; uvsY protein 70 ng/. Mu.L; DNA polymerase 90 ng/. Mu.L; exonuclease 85 ng/. Mu.L; 120nM of target gene probe; target gene forward primer 420nM; target gene reverse primer 420nM; 60nM of internal reference probe sequence; 200nM of reference gene forward primer; 200nM of reference gene reverse primer.
And (3) carrying out negative pressure freeze drying on the prepared amplification system in a freeze dryer to obtain a powder-like amplification system. Can be used in the prior art.
4. HPV6 and HPV11 detection
To the centrifuge tube, polyethylene glycol having a final concentration of 6% (w/v) and a molecular weight of 35000 was added as a reaction buffer to redissolve the system to 48. Mu.L. Adding 2 mu L of DNA of the HPV6 or HPV11 live virus sample extracted in the step 1, wherein the nucleotide sequence of the HPV6 target gene is shown as SEQ ID No. 8; the nucleotide sequence of the target gene of HPV11 is shown as SEQ ID No. 16; the nucleotide sequence of the internal reference DNA is shown as SEQ ID No. 20. Uniformly mixing for 4min by using an even-temperature oscillation mixing instrument of an odd-day instrument, performing instantaneous centrifugation, putting into an instrument capable of detecting FAM and HEX fluorescence, and reacting for 20min at 39 ℃. (Note: to ensure test accuracy, a system without a template was set as a negative control). HPV6 and HPV11 were detected according to the above conditions, each test was repeated twice, and the amplification results of HPV6 are shown in FIGS. 3A and 3B; the amplification results of HPV11 are shown in FIGS. 4A and 4B. As can be seen from FIGS. 3A and 3B, HPV6 group showed amplified fluorescent signal after 1min, and the same amplified signal was obtained by two repeated experiments with good reproducibility. As can be seen from FIGS. 4A and 4B, HPV11 group showed amplified fluorescent signal after 1min, and the same amplified signal was obtained by two repeated experiments, with good reproducibility.
Example 3 internal control-containing double isothermal nucleic acid amplification method for detecting HPV6 and HPV11 viruses according to the present invention
The procedure is as in example 2, except that in 50. Mu.L of the isothermal nucleic acid amplification system, the concentrations of the forward and reverse primers of the target gene are 300nM, respectively, and the other parameters and steps are the same as in example 2. The assay was repeated twice and the amplification results of HPV6 are shown in fig. 5A and 5B; the amplification results of HPV11 are shown in FIGS. 6A and 6B. As can be seen from FIGS. 5A and 5B, HPV6 group started to show amplified fluorescent signal after 1min, except that the peak of the target sequence amplification curve was slightly lower, the results of the two replicates were identical, and the reproducibility was good. As can be seen from FIGS. 6A and 6B, HPV11 group showed amplified fluorescent signal after 1min, and the results of the two replicates were identical and the reproducibility was good. As can be seen from the above, the present invention does not affect the amplification of the target gene at different concentrations of the target gene primer.
Example 4 sensitivity, specificity and detection Limit evaluation of the kit of the present invention
1. Sensitivity evaluation
Serial 10-fold dilution of target gene positive plasmid DNA with concentration range of 10-10 5 Copy/. Mu.L, RNaseP positive plasmid DNA was serially diluted 10-fold at a concentration ranging from 10 to 10 5 Copy/. Mu.L, amplified with serial dilutions of DNA template according to the method of example 2, while detecting different concentrations of target gene and reference plasmid. Wherein the target gene positive plasmid DNA is a plasmid synthesized by taking pUC57 as a vector, and is prepared by Beijing Qinke biological Co., ltd; the RNaseP positive plasmid DNA is a plasmid synthesized by using the internal reference gene sequence pUC57 as a vector, and is prepared by Beijing Optimago Corp.
FIGS. 7A and 7B are graphs showing real-time fluorescence detection amplification of HPV6 and reference DNA at a reference concentration of 100 copies/. Mu.L. Wherein, FIG. 7A is a graph of positive amplified fluorescent signals of HPV6 at different concentrations; FIG. 7B is a graph of amplified fluorescent signals of reference DNA. Each test was repeated 8 times, and the sensitivity of the target gene of HPV6 of the present invention was calculated to be 10 copies/. Mu.L and the sensitivity of the reference gene was calculated to be 100 copies/. Mu.L.
FIGS. 8A and 8B show the case where the internal reference concentration is 10 5 Real-time fluorescence detection amplification plots of HPV6 and internal reference DNA at copy/. Mu.L. Wherein, FIG. 8A is a graph of positive amplified fluorescent signals of HPV6 at different concentrations; FIG. 8B is a graph of amplified fluorescent signals of reference DNA. As can be seen from FIGS. 8A and 8B, at the time of adding 10 5 The copy/. Mu.L of the high concentration reference does not affect the target gene amplification.
FIGS. 9A and 9B are graphs showing real-time fluorescence detection amplification of HPV11 and reference DNA at a reference concentration of 100 copies/. Mu.L. FIG. 9A is a graph showing positive amplified fluorescent signals of HPV11 at different concentrations; FIG. 9B is a graph of amplified fluorescent signals of reference DNA. Each test was repeated 8 times, and the sensitivity of the target gene of the detection method of the present invention was calculated to be 100 copies/. Mu.L, and the sensitivity of the reference gene was calculated to be 100 copies/. Mu.L.
FIGS. 10A and 10B show the case where the internal reference concentration is 10 5 Real-time fluorescence detection amplification plots of HPV11 and internal reference DNA at copy/. Mu.L. FIG. 10A is a graph showing positive amplified fluorescent signals of HPV11 at different concentrations; FIG. 10B is a graph of amplified fluorescent signals of reference DNA. As can be seen from FIGS. 10A and 10B, at the time of addition 10 5 The copy/. Mu.L of the high concentration reference does not affect the target gene amplification.
2. Specificity evaluation
HPV6 and other types of HPVs, including HPV11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 42, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 56, HPV 58, HPV 59, HPV 66, HPV 68 and HPV 81 were detected using the primer set of example 1-1, respectively, and the results are shown in FIGS. 11A and 11B. FIG. 11A is a graph of amplified fluorescent signals of HPV6 and other human papillomaviruses, wherein 1 is an HPV6 amplification curve; 2 is the amplification curve of other human papillomaviruses; FIG. 11B shows the amplification fluorescent signal of reference DNA corresponding to different viruses. The detection result shows that the invention can only specifically detect HPV6 virus and does not cross react with other viruses.
HPV11 and other types of HPVs, including HPV6, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 42, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 56, HPV 58, HPV 59, HPV 66, HPV 68 and HPV 81 were detected using the primer sets of examples 1-2, respectively, and the results are shown in FIGS. 12A and 12B. FIG. 12A is a graph of amplified fluorescent signals of HPV11 and other human papillomaviruses, wherein 1 is an HPV11 amplification curve; 2 is the amplification curve of other human papillomaviruses; FIG. 12B shows the amplified fluorescent signal patterns of reference DNA corresponding to different viruses. The detection result shows that the invention can only specifically detect HPV11 virus and does not cross react with other viruses.
The results of the embodiment show that the kit prepared from the primer probe composition provided by the invention can rapidly detect HPV6 and/or HPV11 viruses at normal temperature, has high sensitivity and good specificity, is easy to popularize and apply in a large range, and has wide market prospect and large economic and social benefits.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Sequence listing
<110> Chinese disease prevention control center viral disease prevention control institute
<120> primer probe combination, kit and application for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or 11
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aggctggtga ctgcccgccc ttagaactta ttaccagtgt tatacaggat ggcgatatgg 180
ttgacacagg ctttggtgct atgaattttg ctgatttgca gaccaataaa tcagatgttc 240
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Claims (10)
1. A set of primer probe combinations for isothermal nucleic acid amplification detection of human papillomavirus type 6 and/or 11, characterized by comprising a first primer probe set and/or a second primer probe set; the first primer probe group comprises a specific primer pair aiming at HPV6 and a target gene probe aiming at HPV 6; the second primer probe set comprises a specific primer pair for HPV11 and a target gene probe for HPV 11;
the specific primer pair for HPV6 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 2; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 3; the target gene probe for HPV6 comprises a nucleotide sequence shown in SEQ ID No. 1;
the specific primer pair for HPV11 comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence shown as SEQ ID No. 10; the reverse primer comprises a nucleotide sequence shown as SEQ ID No. 11; the target gene probe for HPV11 comprises a nucleotide sequence shown in SEQ ID No. 9.
2. The primer probe combination of claim 1, wherein the target gene of the specific primer pair for HPV6 comprises a nucleotide sequence set forth in SEQ ID No. 8; the target gene of the specific primer pair for HPV11 comprises a nucleotide sequence shown as SEQ ID No. 16.
3. The primer probe combination of claim 1, further comprising an endogenous set of internal reference primer probes; the endogenous reference primer probe group comprises an endogenous reference probe and an endogenous reference primer pair; the endogenous reference probe comprises a nucleotide sequence shown as SEQ ID No. 17; the forward primer of the endogenous internal reference primer pair comprises a nucleotide sequence shown as SEQ ID No. 18; the reverse primer of the endogenous reference primer pair comprises a nucleotide sequence shown as SEQ ID No. 19.
4. The primer probe combination of claim 3, wherein the target gene of the endogenous reference primer pair comprises a nucleotide sequence as set forth in SEQ ID No. 20.
5. The primer probe combination of claim 3, wherein the type of endogenous reference probe comprises an exo probe.
6. Use of a primer probe combination according to any one of claims 1 to 5 for the preparation of a tool for detecting human papillomavirus type 6 and/or type 11.
7. A kit for detecting human papillomavirus type 6 and/or 11 comprising the primer probe combination of any one of claims 1 to 5.
8. The kit according to claim 7, wherein the target gene probe for HPV6, the target gene probe for HPV11 and the endogenous reference probe are each labeled with a fluorescent group having different fluorescence; the fluorescent group includes FAM, HEX, ROX, TET, JOE, CY, CY5, TAMRA or VIC.
9. The kit according to claim 7, wherein the isothermal amplification temperature of the kit is 39-42 ℃; the isothermal amplification time of the kit is 15-30 min.
10. Use of the kit according to any one of claims 7 to 9 for the detection of human papillomavirus type 6 and/or type 11 for non-diagnostic purposes, characterized in that the first primer probe set and the endogenous internal reference primer probe set are used in combination when HPV6 is detected; when detecting HPV11, combining the second primer probe group with the endogenous internal reference primer probe group; when HPV6 and HPV11 are detected simultaneously, the first primer probe set and the second primer probe set are combined, and meanwhile, an endogenous internal reference primer probe set is independently provided with a tube to serve as an internal reference.
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| WO2008074182A1 (en) * | 2006-12-18 | 2008-06-26 | Shanghai Tellgen Life Science Co., Ltd. | Method for the detection and typing of human papillomavirus, and the reagent kit thereof |
| CN111676326A (en) * | 2020-07-07 | 2020-09-18 | 郑州安图生物工程股份有限公司 | Detection marker and detection reagent for human papillomavirus type 6 and/or 11 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008074182A1 (en) * | 2006-12-18 | 2008-06-26 | Shanghai Tellgen Life Science Co., Ltd. | Method for the detection and typing of human papillomavirus, and the reagent kit thereof |
| CN111676326A (en) * | 2020-07-07 | 2020-09-18 | 郑州安图生物工程股份有限公司 | Detection marker and detection reagent for human papillomavirus type 6 and/or 11 |
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| Title |
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| 人乳头瘤病毒芯片寡核苷酸探针的研究;危敏;马文丽;李凌;张宝;郑文岭;;微生物学通报(02);100-105 * |
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