CN114107560A - LAMP primer combination, reaction system and method for detecting 16-type and 18-type HPV - Google Patents
LAMP primer combination, reaction system and method for detecting 16-type and 18-type HPV Download PDFInfo
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Abstract
The invention discloses an LAMP primer combination, a reaction system and a method for detecting 16-type and 18-type HPV, which comprise an outer primer, an inner primer and a loop primer. The primer combination, the reaction system and the method provided by the invention are used for detecting nucleic acids of HPV16 and HPV18, SYBR Green dye is adopted for carrying out fluorescent PCR instrument detection and visual dyeing color development detection, the effective amplification and detection of a target gene can be realized under the condition of 55-65 ℃, and temperature changing and complex instruments are not needed; the detection result can be obtained quickly, the reaction can be completed within 30min, the specificity is 100%, and the detection sensitivity is 10-100 copies/mL. The kit is simple, rapid and sensitive to operate, low in hardware requirement and low in cost, is beneficial to being prepared into a kit for wide popularization, provides an effective technical means for field rapid detection and screening of HPV16 and HPV18, and has important significance.
Description
Technical Field
The invention belongs to the field of virus molecular biology, and particularly relates to a LAMP primer combination, a reaction system and a method for detecting 16-type and 18-type HPV.
Background
Cervical cancer is the second leading cause of cancer death in women worldwide. Long-term persistent infection with high-risk genotype Human Papillomavirus (HPV) is a key factor for the subsequent development of pre-cervical lesions. HPV comprises a group of small heterogeneous, non-enveloped capsid-enveloped dsDNA viruses that infect mucosal and cutaneous epithelial cells. The HPV genotypes currently considered to be high-risk types leading to severe cervical dysplasia and cervical cancer development are HPV16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59 and 68, while HPV 26, 53, 66, 73 and 82 are defined as medium-risk types, with HPV16 and HPV18 being the most oncogenic genotypes accounting for about 55.2% and 14.2% of worldwide cervical cancer cases, respectively.
Cervical cancer screening is traditionally based on conventional liquid-based cytological detection (papanicolaou staining) diagnosis. Since liquid-based cytology assays rely heavily on morphological criteria, which is considered to be a highly subjective method, liquid-based cytology screening shows varying degrees of sensitivity (30-87%) at various levels of hospitals. Nucleic acid detection of HPV DNA is now considered to be the gold standard for detection of identified HPV viruses. Because the viral genome is detected in 99.7% of cervical cancer study cases and has higher sensitivity than the traditional liquid-based cytology screening test. HPV genotyping techniques, such as Hybrid Capture 2(HC2), Cervista, Amplicor and Polymerase Chain Reaction (PCR), although they vary in their methods, have the disadvantage of being time consuming and requiring expensive instrumentation. Therefore, it is important to develop a simple, fast and cost-effective method for early detection of HPV DNA.
Loop-mediated isothermal amplification (LAMP) is an increasingly popular alternative for isothermal amplification of nucleotide targets. The LAMP reaction is based on strand displacement amplification with Bst DNA polymerase under isothermal conditions at a temperature range of 60 ℃ to 70 ℃, which requires a set of four (or six) different primers to bind to six (or eight) different regions on the target gene for detection. The set of Lamp primers consists of two outer primers (forward outer primer F3 and reverse outer primer B3), two inner primers (forward inner primer FIP and reverse inner primer BIP) and finally two loop primers (loop forward primer LF and loop back primer LB), and this design can further shorten the amplification time. Thus, at least one set of four primers can be used, which strictly recognize six different regions on the target sequence, ranging from 180bp to 200bp, ensuring high specificity. The LAMP method can produce a large amount of amplification products in the detection of positive samples, and can be detected by agarose gel electrophoresis or by visual color change. The LAMP reaction can be completed within 30-60 minutes, so that any laboratory can carry out nucleic acid detection only by a simple and cheap instrument (such as a water bath or a metal bath). The main advantages of LAMP are: minimal equipment, high sensitivity and rapid detection times (less than 60 minutes).
Disclosure of Invention
The invention aims to provide an LAMP primer combination, a reaction system and a method for rapidly detecting 16-type and 18-type HPV.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, a LAMP detection primer set is provided, which comprises a primer set of HPV16 and/or HPV 18;
the nucleic acid sequence of the primer set of HPV16 is as follows:
an outer primer:
F3：TGTAACCTTTTGTTGCAAGTGT(SEQ ID NO.1)；
B3：AGCATCCCCTGTTTTTTTTTCCA(SEQ ID NO.2)；
an inner primer:
FIP：CAGATGGGGCACACAATTCCTA-CAAAGCACACACGTAGACA(SEQ ID NO.3)；
BIP：ACCATAATCTACCATGGCTG-AACCATCCATTACATCCCGTACC(SEQ ID NO.4)；
loop primer:
LF：GTGCCCATTAACAGGTCTTCC(SEQ ID NO.5)；
LB：CAGGTACCAATGGGGAAGAG(SEQ ID NO.6)；
the nucleic acid sequence of the primer set of HPV18 is as follows:
an outer primer:
F3：AGCGACTCAGAGGAAGAAAAC(SEQ ID NO.7)；
B3：GCCATTGTTGCTTACTGCTG(SEQ ID NO.8)；
an inner primer:
FIP：TCTGGCTTCACACTTACTTCACATA-GTTAATCATCAACATTTACCAG(SEQ ID NO.9)；
BIP：GCTAGTAGTAGAAAGCTCAGCAG-ACCACGGACACACAAAGG(SEQ ID NO.10)；
loop primer:
LF：TTGTGTGACGTTGTGGTTCGGC(SEQ ID NO.11)；
LB：TTCGAGCATTCCAGCAGCTGTT(SEQ ID NO.12)。
in some embodiments of the present invention, the molar ratio of the outer primer, the inner primer and the loop primer in the reaction system is 1: (1-2): 1.
in some preferred embodiments of the present invention, the molar ratio of the outer primer, the inner primer and the loop primer in the reaction system is 1:2: 1.
In a second aspect of the present invention, there is provided a kit comprising the LAMP detection primer set according to the first aspect of the present invention.
In some embodiments of the invention, the kit further comprises Buffer, DNA polymerase, and an indicator.
In some embodiments of the invention, the Buffer comprises: 180-220 mM Tris-Cl pH8.6-9.0, 90-110 mM KCl, 70-90 mM MgSO4、90～110mM(NH4)2SO40.9-1.1% Tween20, 7-9M betaine, 12-16 mM dNTPs.
In some embodiments of the invention, the Buffer comprises: 200mM Tris-Cl pH8.8, 100mM KCl, 80mM MgSO4、100mM(NH4)2 SO 41% Tween20, 8M betaine, 14mM dNTPs.
In some embodiments of the invention, the DNA polymerase is Bst DNA polymerase.
In some embodiments of the invention, the indicator is a fluorescent dye.
In some embodiments of the invention, the fluorescent dye is preferably SYBR Green.
In a third aspect of the invention, there is provided a primer set according to the first aspect of the invention or a kit according to the second aspect of the invention for use in detecting HPV16 and/or HPV 18.
In a fourth aspect of the invention, there is provided a method of detecting HPV16 and/or HPV18, comprising the steps of:
s1: extracting DNA of a sample to be detected;
s2: amplifying a sample DNA using the primer set according to the first aspect of the present invention or the kit according to the second aspect of the present invention;
s3: judging a result according to the indicating condition of the indicator;
this method is not used for the diagnosis of disease.
In some embodiments of the present invention, the amplification condition is a reaction at 55-65 ℃ for 20-30 min.
In some embodiments of the invention, the amplification conditions are a reaction at 60 ℃ for 25 min.
In some embodiments of the invention, the reaction system for amplification is: 2.5. mu.l Buffer, 10pmol F3, 10pmol B3, 20pmol FIP, 20pmol BIP, 10pmol LF, 10pmol LB, 8U Bst DNA polymerase, 1. mu.l template sample DNA, 1X SYBR Green, plus ddH2O to 25. mu.l.
The invention has the beneficial effects that:
the primer combination, the reaction system and the method provided by the invention are used for detecting nucleic acids of HPV16 and HPV18, SYBR Green dye is adopted for carrying out fluorescent PCR instrument detection and visual dyeing color development detection, the effective amplification and detection of a target gene can be realized under the condition of 55-65 ℃, the temperature change is not needed, and complex instruments are not needed; and the detection result can be rapidly obtained, the reaction can be completed within 30min, the specificity is 100%, and the detection sensitivity is 10-100 copies/mL. The kit is simple, rapid and sensitive to operate, low in hardware requirement and low in cost, is beneficial to technical preparation into a kit for wide popularization, provides an effective technical means for field rapid detection and screening of HPV16 and HPV18, and has important significance.
Drawings
FIG. 1 shows the results of fluorescence curves for detection of HPV16 and HPV 18.
FIG. 2 is a visualization of the detection of HPV16 and HPV 18.
FIG. 3 shows the result of HPV16 detection sensitivity experiment.
FIG. 4 shows the result of HPV18 detection sensitivity experiment.
FIG. 5 shows the result of HPV16 detection-specific experiment.
FIG. 6 shows the result of HPV18 detection-specific experiment.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1 primer design, reaction System and detection method for LAMP detection of HPV16 and 18
1. Conserved sequences of HPV16 and HPV18 are selected to design corresponding LAMP primers, wherein the LAMP primers comprise two outer primers (F3 and B3), two inner primers (FIP and BIP) and corresponding loop primer pairs LF and LB. Primer synthesis was synthesized by Biotechnology engineering (Shanghai) Inc., and purified by HPLC. The primer information is shown in Table 1.
TABLE 1 primer information
F3 is a primer having an F3 region complementary to the F3c region of the target gene.
B3 is a primer having a B3 region interacting with the B3c region of the target gene.
FIP is a primer having a F2 region complementary to the F2c region of the target sequence at the 3-terminus and a sequence identical to the F1c region of the target gene at the 5-terminus.
BIP is a primer having a B2 region complementary to the region of the target sequence B2c at the 3 'end and the same sequence as the Blc region of the target gene at the 5' end.
LF is a primer of LF region complementary to the loopF region of the target gene.
LB is a primer having an LB region complementary to the loopB region of the target gene.
2. A reaction system comprises a primer group for detecting HPV16 and HPV18, and specifically comprises the following components, and the reaction system is shown in Table 2:
TABLE 2 reaction System
The 10X Buffer comprises the following components:
3. a kit, comprising the reaction system, for detecting HPV16 and 18.
Example 2A method for LAMP detection of HPV16 and 18
In this example, a method for detecting HPV16 and 18 using LAMP, comprising the steps of:
(1) extracting DNA of a sample to be detected by adopting a genome DNA extraction kit;
(2) adding DNA of a sample to be detected into a reaction system, placing the reaction system on a fluorescent PCR instrument, setting the temperature at 60 ℃ for 1 minute, and carrying out LAMP reaction for 25 cycles;
(3) collecting fluorescent signals by a fluorescent PCR instrument;
(4) visualization of the reaction was performed using 1. mu.l SYBR Green.
The results are shown in FIG. 1 for fluorescence signal results and in FIG. 2 for visualization. The result shows that the LAMP primer combination and the reaction system of the 16-type and 18-type HPV can detect the 16-type and 18-type HPV positive samples.
Example 3 sensitivity detection
Using human papillomavirus deoxyribonucleic acid (HPV DNA) standards (bang de sheng biotechnology, guang) as positive samples, the average concentration of HPV16 was 1.36E +06 and the average concentration of HPV18 was 2.13E + 06. Diluting to 10 with deionized water or TE Buffer6、105、104、103、102、101Copies/. mu.l were used as standards and are designated 1, 2, 3, 4, 5 and 6, respectively. LAMP detection is carried out on the standard substances with different concentrations, the reaction system and the reaction conditions are the same as those in example 1, and the results are shown in fig. 3 and fig. 4, and the results show that the detection sensitivity of HPV16 and 18 is 10-100 copies.
Example 4 specific assay
DNA samples which are clinically used for determining HPV infection subtypes by a fluorescent probe PCR method and comprise 16, 18, 31, 33 and 52 types of positive samples are selected, the DNA of a sample to be detected is extracted by adopting a genome DNA extraction kit, 20ng of DNA (about 3000 copies of human genome DNA) of the sample to be detected is taken as a template for LAMP detection, and the reaction system and the reaction conditions are the same as those in example 1. The results are shown in FIGS. 5 and 6.
The result shows that the negative DNA of HPV16 and 18 type and the positive DNA of non-HPV 16 and 18 type have no positive result, and the positive DNA of HPV16 and 18 type shows positive result, which shows that the primer group of the embodiment 1 of the invention has strong specificity.
The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Guangzhou Lankun Biotechnology GmbH
<120> LAMP primer combination, reaction system and method for detecting 16-type and 18-type HPV
<130>
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Claims (10)
1. An LAMP detection primer set comprising a primer set of HPV16 and/or HPV 18;
the nucleic acid sequence of the primer set of HPV16 is as follows:
an outer primer:
F3：TGTAACCTTTTGTTGCAAGTGT(SEQ ID NO.1)；
B3：AGCATCCCCTGTTTTTTTTTCCA(SEQ ID NO.2)；
an inner primer:
FIP：CAGATGGGGCACACAATTCCTA-CAAAGCACACACGTAGACA(SEQ ID NO.3)；
BIP：ACCATAATCTACCATGGCTG-AACCATCCATTACATCCCGTACC(SEQ ID NO.4)；
loop primer:
LF：GTGCCCATTAACAGGTCTTCC(SEQ ID NO.5)；
LB：CAGGTACCAATGGGGAAGAG(SEQ ID NO.6)；
the nucleic acid sequence of the primer set of HPV18 is as follows:
an outer primer:
F3：AGCGACTCAGAGGAAGAAAAC(SEQ ID NO.7)；
B3：GCCATTGTTGCTTACTGCTG(SEQ ID NO.8)；
an inner primer:
FIP：TCTGGCTTCACACTTACTTCACATA-GTTAATCATCAACATTTACCAG(SEQ ID NO.9)；
and (3) BIP: GCTAGTAGTAGAAAGCTCAGCAG-ACCACGGACACACAAAGG (SEQ ID NO. 10); loop primer:
LF：TTGTGTGACGTTGTGGTTCGGC(SEQ ID NO.11)；
LB：TTCGAGCATTCCAGCAGCTGTT(SEQ ID NO.12)。
2. the primer set according to claim 1, wherein the molar ratio of the outer primer, the inner primer and the loop primer in the reaction system is 1: (1-2): 1.
3. a kit comprising the LAMP detection primer set according to claim 1 or 2.
4. The kit of claim 3, wherein the kit further comprises Buffer, DNA polymerase, and an indicator.
5. The kit according to claim 4, wherein the Buffer comprises: 180 to 220mM Tris-ClpH8.6～9.0、90～110mM KCl、70～90mM MgSO4、90～110mM(NH4)2SO40.9-1.1% Tween20, 7-9M betaine, 12-16 mM dNTPs.
6. The kit of claim 4, wherein the DNA polymerase is Bst DNA polymerase.
7. The kit of claim 4, wherein the indicator is a fluorescent dye; the fluorescent dye is preferably SYBR Green.
8. Use of the primer set of claim 1 or 2 or the kit of any one of claims 3 to 7 for detecting HPV16 and/or HPV 18.
9. A method of detecting HPV16 and/or HPV18, comprising the steps of:
s1: extracting DNA of a sample to be detected;
s2: amplifying a sample DNA using the primer set of claim 1 or 2 or the LAMP kit of any one of 3 to 7;
s3: judging a result according to the indicating condition of the indicator;
this method is not used for the diagnosis of disease.
10. The method of claim 9, wherein the amplification conditions are: reacting for 10-30 min at 55-65 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805218A (en) * | 2015-04-02 | 2015-07-29 | 赣南医学院第一附属医院 | LAMP-and-molecular-beacon-based reaction system and method for detecting HPV16 and HPV18 |
| CN107557492A (en) * | 2017-09-14 | 2018-01-09 | 温州美众医学检验所 | LAMP detections are combined with primer and its reaction system |
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| CN104805218A (en) * | 2015-04-02 | 2015-07-29 | 赣南医学院第一附属医院 | LAMP-and-molecular-beacon-based reaction system and method for detecting HPV16 and HPV18 |
| CN107557492A (en) * | 2017-09-14 | 2018-01-09 | 温州美众医学检验所 | LAMP detections are combined with primer and its reaction system |
| US20230002839A1 (en) * | 2019-09-09 | 2023-01-05 | Genomtec S.A. | Primer sets for the detection of human papillomavirus type 16 (hpv16) and human papillomavirus type 18 (hpv18), the method of detecting hpv16 and hpv18 infections, the use of a primer set for the detection of hpv16 and hpv18 infections |
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