CN110923362A - Colloidal gold chromatography kit for simultaneously detecting herpes simplex virus I/II and application thereof - Google Patents
Colloidal gold chromatography kit for simultaneously detecting herpes simplex virus I/II and application thereof Download PDFInfo
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Abstract
The invention discloses a colloidal gold chromatography kit for simultaneously detecting herpes simplex virus I/II and application thereof. The kit realizes the amplification of pathogen nucleic acid fragments through the steps of cracking collected samples by cell lysate to release pathogen nucleic acid, and then performing reverse transcription and transcription under the action of reverse transcriptase and T7RNA polymerase. The amplified RNA product is identified and captured by a specific probe in the detection liquid to form an RNA amplification product-specific probe-gold probe compound, and the compound is fixed on an NC membrane through lateral flow chromatography to form a visible strip, so that the detection of pathogen nucleic acid is realized. The invention has no RNA extraction process, does not need special instruments, is based on RNA isothermal amplification, is not easy to pollute in actual detection, has the advantages of high sensitivity, strong specificity and simple operation, and makes the wide application of herpes simplex virus I and II nucleic acid detection possible.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for detecting herpes simplex virus I/II nucleic acid based on RNA isothermal amplification-gold probe chromatography technology and application thereof.
Background
Herpes Simplex Virus (HSV) is a viral pathogen that seriously harms human health and causes skin and venereal diseases. HSV is transmitted primarily by direct contact, with about 80% of those infected clinically presenting as asymptomatic infections, a major cause of the HSV epidemic. HSV can be divided into two serotypes, namely HSVI and HSVII, according to different antigenicity. HSVI mainly causes infection of the skin mucosa of eyes and mouths and the central nervous system above the waist, has light symptoms and is easy to treat; HSVII mainly causes infection of skin mucosa and newborn baby of genital part, and pregnant women infected with HSVII can induce abnormal fetal development or abortion. Because HSVII type infection is easy to recur, the medication period is longer than that of HSVI infection. Therefore, it is necessary to perform typing detection to avoid drug abuse.
There are many laboratory diagnostic methods for HSV infection, which can be generally classified as direct smear, virus isolation and culture, serological detection, and PCR. The direct smear method has the advantages of quick detection, low cost and visual result, but the detection result is easily influenced by the sampling position and the pathological change time. The virus isolation culture is the gold standard for HSV detection, and although the method is reliable, the method has high requirement on experimental conditions, takes long time (2 to 3 weeks), is easy to pollute, and therefore, the method cannot be effectively applied clinically. The serological detection mainly adopts an ELISA method to detect the antigen or the antibody in the serum, the method has moderate specificity and sensitivity, is simple, convenient and quick, is also a detection method commonly used in clinic at present, but has the problem of false negative and false positive which is difficult to solve. The PCR method can directly detect HSV nucleic acid, has high sensitivity, strong specificity and higher detection speed, has considerable advantages in the aspects of shortening the detection window period and improving the pathogen detection rate, but has certain requirements on hardware facilities, needs special PCR diagnosis laboratories and expensive experimental instruments, and is not beneficial to popularization and application in some communities and remote hospitals. Therefore, there is still a need to find a simple, rapid and inexpensive diagnostic method for herpes simplex virus.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a kit for detecting herpes simplex virus type I/II nucleic acid based on RNA isothermal amplification-gold probe chromatography and its application. The kit realizes the amplification of pathogen nucleic acid fragments through the steps of cracking collected samples by cell lysate to release pathogen nucleic acid, and then performing reverse transcription and transcription under the action of reverse transcriptase and T7RNA polymerase. The amplified RNA product is identified and captured by a specific probe in the detection liquid to form an RNA amplification product-specific probe-gold probe compound, and the compound is fixed on an NC membrane through lateral flow chromatography to form a visible strip, so that the detection of pathogen nucleic acid is realized. Therefore, the invention has no complex RNA extraction process, does not need special instruments, is not easy to pollute in actual detection based on the characteristic that RNA molecules are easy to degrade, has the advantages of high sensitivity, strong specificity and simple operation, and makes the wide application of herpes simplex virus I/II nucleic acid detection possible.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, a colloidal gold chromatography kit for joint detection of herpes simplex virus type I/type II nucleic acid is provided, the kit is based on RNA isothermal amplification-gold probe chromatography technology, and comprises:
1) amplification reaction solution: containing 40mM Tris-HCl (pH 8.0), 12mM MgCl270mM KCl, 15% DMSO, 5mM DTT, 1mM of each dNTP, 2mM of each NTP, and 0.2. mu.M of amplification primer; wherein the amplification primers comprise three pairs: the gene comprises I type herpes simplex virus, II type herpes simplex virus and human reference genes, and specifically comprises the following genes:
(1) type I herpes simplex virus (UL30 gene a conserved region sequence) amplification primers:
HSVI-F(5’-3’)：CGTCGGGTCGGGCGGCTTCT；
HSVI-R(5’-3’)：TAATACGACTCACTATAGGGAGAGGTGTGATGGCGTCCATAAA；
(2) type II herpes simplex virus (UL30 gene a conserved region sequence) amplification primers:
HSVII-F(5’-3’)：CCACCGTCACCGTCTTCCAC；
HSVII-R(5’-3’)：AATACGACTCACTATAGGGAGAACCTCCGCCTTGTTCATGTAAA；
(3) amplification primers for the reference gene (a conserved region sequence of the human 18 SRRNA):
18S-F(5’-3’)：AGAAACGGCTACCACATCC；
18S-R(5’-3’)：TAATACGACTCACTATAGGGAGACACCAGACTTGCCCTCCA；
as the homology of the I type herpes simplex virus sequence and the II type herpes simplex virus sequence is as high as about 50 percent, sequences with higher sequence conservation and larger difference between the two types are selected as possible when primers are designed. Ensures that different primers do not interfere with each other while ensuring high amplification efficiency of respective single primers. The 5' ends of the R primers of the three pairs of primers are introduced with a T7RNA polymerase promoter sequence.
2) Amplification enzyme: comprises three kinds, reverse transcriptase (such as AMV or M-MLV), T7RNA polymerase and RNASEH.
3) Cell lysate (purchased from SIGNASIS, USA, under the product number CL-0001): the cells can be lysed, releasing the nucleic acids.
4) Detection liquid: the kit comprises a nucleic acid probe (gold probe) marked by colloidal gold particles, a specific probe of each index and a C-line chromogenic probe, wherein two specific probes of each index are respectively a CES series and an LES series, the CES series and the LES series can be designed into a plurality of probes, and the probes are specifically (5 '-3') as follows:
(1) i type herpes simplex virus specific probe
HSVI-CES1：GGCCGCGGCGCTCGCGCCTTTTTATCTATAGCTGGTGT；
HSVI-CES2：TGTGGGGCGGCGTGGACCTTTTTATCTATAGCTGGTGT；
HSVI-LES1：ACGCCCCGGCGGGGTTCATTTTCGCAGTGCTCGAGCTCTGAGC；
HSVI-LES2：ACCCCACCGTCACCGTCTTTTTCGCAGTGCTCGAGCTCTGAGC；
HSVI-LES3：TTCACGTGTATGACATCCTTTTCGCAGTGCTCGAGCTCTGAGC；
(2) II type herpes simplex virus specific probe
HSVII-CES1:GCGTACAGCATGCGCGCCGCTTTTCTATGTATCTGTGAGT；
HSVII-CES2:CCAGCTCCACGAGCGATTTATTTTCTATGTATCTGTGAGT；
HSVII-LES1:TGGACGCCATCACGCCCGCCTTTTCGCAGTGCTCGAGCTCTGAGC；
HSVII-LES2:GGGACCGTCATCACGCTTCTTTTTCGCAGTGCTCGAGCTCTGAGC；
HSVII-LES3:GGGTCTGACCCCCGAAGGCCTTTTCGCAGTGCTCGAGCTCTGAGC；
(3) Specific probe for internal reference
Internal reference (CES 1: AAGGAAGGCAGCAGGCTTTTATCTGTATAGTGTCTG);
internal reference (CES 2: GCGCAAATTACCCACTTTTTATCTGTATAGTGTCTG);
internal reference LES1: CCCGACCCGGGGAGGTTTTTCGCAGTGCTCGAGCTCTGAGC;
internal reference LES2: AGTGACGAAAAATAACTTTTCGCAGTGCTCGAGCTCTGAGC;
internal reference LES3: AATACAGGACTCTTTCTTTTCCGCAGTGCTCGAGCTCTGAGC;
(4) c line color probe (5 '-3')
TCAGATCACTATGTACTTTTCGCAGTGCTCGAGCTCTGAGC；
(5) Gold probe
The 5' end of the gold probe is modified by sulfhydrylation, and the sequence is as follows:
5’-CCTACTCTGCAGTGCTCCATCGTACGTCTGTCATTTTTGCTCAGAGCTCGAGCACTGCG-3’；
5) the test paper strip: the test strip is fixed on a PVC bottom plate, and a sample pad, an NC membrane and absorbent paper are arranged from left to right in sequence; the NC film is provided with a C line (quality control line) and three T lines (detection lines), and the directions from the sample pad to the absorbent paper are respectively HSVII-T, HSVI-T, reference-T and C lines (as shown in figure 3); an HSVI coated probe is coated at an HSVI-T position, an HSVII-T coated probe is coated at an HSVII-T position, an internal reference coated probe is coated at an internal reference-T position, and a C line coated probe is coated at a C line, wherein the specific sequence (5 '-3') is as follows:
c, coating of a probe: GTACATAGTGATCTGATTTTGTACATAGTGATCTGA, respectively;
coating a probe with an internal reference line: CAGACACTATACAGATTTTTCAGACACTATACAGAT, respectively;
HSVI coil-coated probe: ACACCAGCTATAGATATTTTACACCAGCTATAGATA, respectively;
HSVII coil-coated probes: ACTCACAGATACATAGTTTTACTCACAGATACATAG are provided.
The invention provides a method for detecting herpes simplex virus I/II type nucleic acid by using the kit for detecting herpes simplex virus I/II type nucleic acid based on RNA isothermal amplification-gold probe chromatography technology, which comprises the following steps:
(1) isothermal amplification of RNA
The detection indexes of the invention are three: herpes simplex virus type I, herpes simplex virus type II and human reference gene. A pair of (F/R primers) amplification primers is designed for each index, wherein the 5' end of the R primer carries a T7RNA polymerase promoter. The invention realizes the amplification of each index nucleic acid in the same amplification tube, and the specific steps are as follows: during amplification, under the action of an R primer with a T7 promoter and reverse transcriptase, converting RNA to be detected into an RNA-cDNA hybrid; RNA in cDNA is digested by RnaseH in the amplified enzyme to obtain single-stranded cDNA; synthesizing a second strand under the action of the F primer and the DNA polymerase function of reverse transcriptase to form double-stranded DNA with a T7 promoter; the double-stranded DNA with the T7 promoter is transcribed to generate an RNA molecule product under the action of T7RNA polymerase. The transcribed RNA molecule product can enter a circulating amplification process, firstly, the F primer is combined with the transcribed RNA molecule product, and the transcribed RNA is converted into an RNA-cDNA hybrid under the action of reverse transcriptase; RNA in cDNA is digested by RnaseH in the amplified enzyme to obtain single-stranded cDNA; the R primer will then bind to the single-stranded cDNA, synthesize a second strand under the action of the DNA polymerase function of the reverse transcriptase, enrich and synthesize more double-stranded DNA molecules with the T7 promoter again, provide more transcription templates for the T7RNA polymerase, and further generate a large amount of RNA molecule products under the action of the T7RNA polymerase (see FIG. 1).
The invention designs the detection of the internal reference gene, aiming at monitoring the effectiveness of sample collection and the effectiveness of an amplification system. When the sample is qualified, the sample must contain human exfoliated cells and be detected in the detection process, the internal reference should be positive when the sample is negative, otherwise, the whole detection needs to be re-sampled for retesting.
(2) Gold Probe chromatography
a, designing a specific probe, a gold probe, a C-line color probe and a coated probe
Specific probes: each index-specific probe includes two types: CES series and LES series, multiple probes can be designed for each probe. The CES probe comprises two parts, one end of the CES probe can be specifically combined with an amplified RNA product, the other end of the CES probe can be assembled with a coated probe coated on an NC membrane to play a role in fixing the amplified product RNA, and the two parts are linked by 4-5T. Each LES probe also comprises two parts, one end of each LES probe can be specifically combined with the amplified RNA product, the other end of each LES probe can be combined with the gold probe to play a role in color development of the linked gold probe, and the two parts are linked by 4-5T.
Gold probe: the 5' end of the gold probe is modified by sulfhydrylation, and a sulfhydryl group can form a covalent bond with the colloidal gold particles and is marked on the colloidal gold particles. The gold probe may be bound to one end of a specific probe LES.
Coating the probe: the coated probe is fixed on an NC membrane and can be combined with one end of a specific probe CES to play a role in fixation. Each coated probe contains two copies, which are connected by 4-5T.
C-line color probe: comprises two parts which are linked by 4-5T. One end of the probe can be combined with a gold probe, and the other end of the probe can be combined with a C-coated probe coated on an NC membrane. During chromatography, the C-line chromogenic probe can form a 'C-line chromogenic probe-gold probe' complex no matter whether RNA amplification products exist or not, and the complex can be captured and intercepted by the C-line on an NC membrane by the probe during chromatography to form a strip visible to naked eyes. The probe can control the quality of the test paper strip and the detection liquid, and the chromatography process is error-free.
The specific probe is designed without crossing between different probes with the same index, and CES series are not crossed with a gold probe and a coated probe, so that the detection specificity is ensured.
The CES series and LES series of the specific probes are designed to improve the efficiency of immobilization and to bind more gold probes, thereby improving the sensitivity of detection.
b, detecting by test paper
The test strip is provided with a detection line and a quality control line, wherein the detection line comprises HSVI-T, HSVII-T and internal reference-T, wherein an HSVI coated probe coated at the HSVI-T can be specifically combined with one end of a CES series probe of the herpes simplex virus I; the HSVII-T coated HSVII-coated probe can be specifically combined with one end of a II type herpes simplex virus CES series probe; the internal reference coated probe coated at the internal reference-T can be specifically combined with one end of the internal reference CES series probe. The C-line coated probe coated on the quality control line (C line) can be specifically combined with the C-line chromogenic probe. And hybridizing the specific probe CES, the specific probe LES, the gold probe and the specific amplification product of the nucleic acid to be detected, and then dripping the hybridized product on a test strip for chromatography, wherein the detection line color development shows that the nucleic acid to be detected exists, and the quality control line color development shows that the detection is effective.
In combination with the above principle, the working process of the above method of the present invention is described as follows:
(1) nucleic acid extraction
Collecting a genital tract or urethra swab sample of a suspected herpes simplex virus patient, and releasing virus nucleic acid molecules by using a cell lysate lysis method.
(2) Isothermal amplification of RNA
mu.L of nucleic acid extract was added to 17. mu.L of amplification reaction solution containing herpes simplex virus type I, herpes simplex virus type II and internal reference primer, heated at 95 ℃ for two minutes, preheated at 42 ℃ for 2 minutes, added with 1. mu.L of amplification enzyme, and amplified at 42 ℃ for 1 hour. If there is herpes simplex virus nucleic acid in the sample to be detected, a large amount of amplification enrichment is carried out on the index RNA molecules during amplification.
(3) Chromatography of test paper strip
a, prehybridization
The RNA isothermal amplification product was mixed with the detection solution (including specific probe, gold probe and C-line color probe) and prehybridized at 42 ℃ for 10 minutes. The amplified RNA molecules are combined with specific probes (including CES series probes and LES series probes) in a complementary pairing mode. One end of CES series probe is hybridized and complemented with RNA molecule, and the other end is combined with coating probe on NC film; one end of the LES series probe is hybridized and complementarily paired with the RNA molecule, the other end can be complementarily paired and combined with the gold probe, and when an amplification product exists, a CES probe-RNA molecule-LES probe-gold probe compound can be formed.
b, chromatographic detection
Dropping the pre-hybridization product at the sample pad of the test strip, carrying out chromatography on the pre-hybridization solution along the NC membrane in the direction of absorbent paper, and when an RNA amplification product to be detected exists, forming a 'CES probe-RNA molecule-LES probe-gold probe complex', and intercepting the complex by a coating probe coated on the NC membrane during chromatography to form a strip which is seen by naked eyes and is positive (as shown in figure 4).
If the RNA product to be detected is not amplified, the CES probe-RNA molecule-LES probe-gold probe compound is not formed, the colloidal gold particles cannot be gathered at the T line, and a macroscopic strip cannot be formed, so that the detection result is negative (shown as negative in figure 4).
The C-line chromogenic probe can form a "C-line chromogenic probe-gold probe" complex no matter whether the RNA product to be detected is amplified or not, the complex flows forwards along an NC membrane during chromatography, and when the C-line chromogenic probe reaches the C-line, the complex is combined with the probe coated at the C-line, so that the complex is retained at the C-line to form a colored band visible to naked eyes, and the experimental result is valid (as shown in fig. 4).
In a second aspect, the application of the kit for detecting the herpes simplex virus I/II nucleic acid based on the RNA isothermal amplification-gold probe chromatography technology in preparation of a herpes simplex virus I and/or II detection reagent is provided.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention can simultaneously amplify three indexes of the I type herpes simplex virus, the II type herpes simplex virus and the reference gene in the same tube by an RNA constant temperature amplification method, the amplified nucleic acid product is RNA, the RNA is easy to degrade in the natural environment, and compared with the DNA amplified by a PCR method, the invention has the effect of preventing pollution more easily. RNA isothermal amplification is carried out in an environment of 42 ℃, and amplification reaction can be realized even in a water bath kettle, so that the requirements of experimental instruments are reduced to the maximum extent.
2. As the homology of the I type herpes simplex virus and the II type herpes simplex virus is as high as about 50 percent, sequences with higher sequence conservation and larger difference between the two types are selected as possible when primers are designed, so that the aim of full coverage and typing detection of the two types of viruses can be fulfilled. Meanwhile, the invention ensures that each single primer has high amplification efficiency, different primers have no interference with each other and the overall amplification effect is good through multi-round tests when designing the primers.
3. The specific probe CES series and the specific probe LES series introduced during design have the function of bridge molecular components, and the gold probe and the RNA nucleic acid amplification fragment are successfully combined in series by the two probes to realize specific detection of the index RNA nucleic acid fragment. By using the two sets of probes, any one set of probes and the index nucleic acid amplification fragment can not be successfully immobilized on an NC membrane when the hybridization fails, so that a positive detection result cannot be generated, and the detection specificity is ensured. The test results of the kit of the invention on 32 other microorganisms listed in Table 3 are negative, which proves that the kit of the invention has no cross reaction with other microorganisms. Wherein each set of probe can be designed into more than two sets, and the design is favorable for improving the sensitivity of the test strip. The kit has the lowest detection limit of 8.0TCID50/mL for HSVI (ATCC VR-539) and the lowest detection limit of 6.4TCID50/mL for HSVII (ATCC VR-540). The detection sensitivity and specificity of 464 diagnosis results of the type I and type II herpes simplex virus clinical samples are higher than those of a certain commercialized fluorescent quantitative PCR kit for detecting the type I and type II herpes simplex virus.
4. The invention adopts RNA isothermal amplification technology and test strip chromatography technology, not only applies the characteristic of low requirement of RNA isothermal amplification on instruments, but also successfully integrates the characteristic of rapidness of colloidal gold. The test paper strip is used for detecting nucleic acid, and the result can be interpreted only in about 10 min. The method is also very simple in operation, has low technical requirements on experimenters, does not need special instruments and equipment, and is easy to popularize the nucleic acid detection of the herpes simplex virus to basic level and remote rural medical institutions.
Drawings
FIG. 1 is a schematic diagram of isothermal amplification of RNA;
FIG. 2 is a schematic diagram of a test strip color development;
FIG. 3 is a schematic view of the test strip;
FIG. 4 is a schematic diagram of detection of negative and positive;
a: HSVI negative, HSVII negative, internal reference negative;
b: HSVI negative, HSVII negative, internal reference positive;
c: HSVI positive, HSVII negative and internal reference positive;
d: HSVI negative, HSVII positive and internal reference positive;
e: positive for HSVI, positive for HSVII and positive for internal reference;
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
The experimental methods in the following examples, in which specific conditions are not specified, are generally performed according to conventional conditions, such as "molecular cloning: the conditions described in the laboratory Manual 3 rd edition (New York: Cold Spring Harbor laboratory Press,2005) were carried out.
[ example 1 ] preparation of test paper for nucleic acid detection
The main raw materials required for preparing the nucleic acid detection test strip are as follows: nitrocellulose membranes (NC membranes), sample pads, absorbent paper, PVC base plates, and the like.
1. Film spraying:
detection line HSVII-T: the kit can capture and bind a CES sequence of an HSVII specific probe, 10 mu M of the HSVII coated probe, and has the following film spraying amount: 2-3 muL/cm;
detection line HSVI-T: can capture and combine a CES sequence of an HSVI specific probe, a 10 mu M HSVI coating probe, and the film spraying amount is as follows: 2-3 muL/cm;
detecting internal reference-T: the probe can capture and combine a CES sequence of an internal reference specific probe, 10 mu M of an internal reference coated probe, and the film spraying amount is as follows: 2-3 muL/cm;
quality control line (line C): can capture and bind to a C line chromogenic probe sequence, a 10 mu M C line coated probe, a film spraying amount: 2-3 muL/cm;
after the film spraying is finished, the film is automatically crosslinked once in an ultraviolet crosslinking instrument, and the film is placed in a clean constant temperature cabinet at 37 ℃ for drying for 2 hours and stored in a dry environment for later use.
2. Test strip assembly
And respectively cutting 2cm long absorbent paper, the coated NC film and the sample pad, and sequentially fixing the absorbent paper, the coated NC film and the sample pad on a PVC base plate from top to bottom to obtain the detection test strip. The structure of the test strip is shown in fig. 3.
[ example 2 ] sensitivity test
Virus stock solutions of HSVI (ATCC No. VR-539) and HSVII (ATCC No. VR-540) from ATCC are subjected to gradient dilution liquid minimum detection limit determination, each gradient virus dilution solution is repeated for 3-5 parts, each part is subjected to repeated detection for 20 times, the virus level with 90% -95% positive detection rate is taken as the minimum detection limit, and the detection results are as follows:
HSVI minimum detection limit detection
TABLE 1.1 data of testing experiments for different titers of HSVI
TABLE 1.2 HSVI minimum detection limit experimental data
HSVII minimum detection Limit detection
TABLE 2.1 data of the test experiments for different titres of HSVII
TABLE 2.2 HSVII minimum detection Limit Experimental data
Finally, the detection sensitivity of the kit is determined as follows:
| detecting the index | Viral strains | Minimum limit of detection |
| HSVI | ATCC VR-539 | 8.0TCID50/mL |
| HSVII | ATCC VR-540 | 6.4TCID50/mL |
[ example 3 ] specificity verification
1, test strains
Different microorganisms are detected after nucleic acid is extracted, and the specificity of the primer and probe design of the kit is verified. The relevant pathogens and titers were as follows:
table 3 specific verification of test strain information
| Microorganisms | Concentration of | Microorganisms | Concentration of |
| Adenovirus type 3 | 1.58×107TCID50/mL | Mumps virus | 6.3×109TCID50/mL |
| Adenovirus type 7 | 1.58×108TCID50/mL | Respiratory syncytial virus type A | 1.58×107TCID50/mL |
| Coxsackievirus B group 5 type | 2.8×108TCID50/mL | Respiratory syncytial virus type B | 8.89×105TCID50/mL |
| Echovirus type 9 | 1.58×107TCID50/mL | Chlamydia pneumoniae | 1.58×108TCID50/mL |
| Enterovirus 71 | 1.58×106TCID50/mL | Escherichia coli | (12～14)×108cfu/mL |
| Coxsackievirus group A16 type | 1.58×105TCID50/mL | Haemophilus influenzae | 2.4×106cfu/mL |
| Human parainfluenza virus type 1 | 1.58×104TCID50/mL | Non-toxic mycobacterium tuberculosis | 3.2×106cfu/mL |
| Human parainfluenza virus type 2 | 2.8×106TCID50/mL | Streptococcus pneumoniae | 2.6×106cfu/mL |
| Human parainfluenza virus type 3 | 1.58×107TCID50/mL | Streptococcus pyogenes | 2.0×106cfu/mL |
| Measles virus | 3.2×109TCID50/mL | Salivary streptococcus | 2.1×106cfu/mL |
| Mumps virus | 8.89×105TCID50/mL | Human coronavirus | 1.6×106TCID50/mL |
| Rubella virus | 3.2×109TCID50/mL | Cytomegalovirus | 3.0×106TCID50/mL |
| Mycoplasma pneumoniae | 106cfu/mL | Rhinovirus type 1A | 4.0×106TCID50/mL |
| Pseudomonas aeruginosa | (14～22)×108cfu/mL | Bordetella pertussis | 5.5×106cfu/mL |
| Staphylococcus aureus | (24～40)×108cfu/mL | Neisseria meningitidis | 6×106cfu/mL |
| Gonococci | 1.0×106cfu/mL | Human metapneumovirus | 2.8×106TCID50/mL |
2, test results
The test results were as follows:
TABLE 4 specificity verification test results
3, conclusion
The data show that the detection results of the kit disclosed by the invention on the microorganisms are negative, so that the kit disclosed by the invention is proved to have no cross reaction with other microorganisms, and the strong specificity of the kit for detecting pathogens is embodied.
Example 4 validation of clinical samples
1, clinical sample information
464 swab samples of the genital tract or the urethra were co-detected in the first hospital, Wuhan City of Hubei province, wherein 176 and 288 samples of male and female specimens, respectively, accounted for 37.93% and 62.07%, respectively. Of the 464 specimens, patients were 67 years old at the maximum and 1 month at the minimum, with a mean age of 28.13 years, a standard deviation of 10.5 years and a median of 30 years. The diagnosis of the patients in the group is related to the herpes simplex virus infection,
2, detecting the result
(1) Detection result of type I herpes simplex virus
When in detection, the kit and a certain commercialized fluorescent PCR kit for detecting the herpes simplex virus I and II are used for simultaneously detecting samples, and detection results are summarized into a four-table as follows:
3 inconsistent samples are retested by adopting a gene sequencing method, 1 sample is positive, and the kit is a commercial detection I type and II type herpes simplex virus fluorescent PCR kit for detecting the positivity of the patent of the invention and is a negative sample. Sequencing results show that one commercial fluorescent PCR kit for detecting the herpes simplex viruses I and II is missed, and 2 false positive samples are obtained, and obviously, the kit disclosed by the invention has higher detection sensitivity and stronger specificity when used for detecting clinical samples.
(2) Detection result of II type herpes simplex virus
When in detection, the kit and a certain commercialized fluorescent PCR kit for detecting the herpes simplex virus I and II are used for simultaneously detecting samples, and detection results are summarized into a four-table as follows:
9 inconsistent samples are retested by adopting a gene sequencing method, 5 samples are positive, and the kit is a commercial detection I type and II type herpes simplex virus fluorescent PCR kit for detecting the positivity of the patent of the invention and is used for detecting negative samples. Sequencing results show that 5 commercial fluorescent PCR kits for detecting I-type and II-type herpes simplex viruses are missed and 4 false positive samples are false positive, and obviously, the kit disclosed by the invention has higher sensitivity and stronger specificity in detection of the II-type herpes simplex viruses when used for detecting clinical samples.
Sequence listing
<110> Wuhan Zhongban Biotechnology GmbH
<120> colloidal gold chromatography kit for simultaneously detecting herpes simplex virus I type/II type and application thereof
<160>27
<170>SIPOSequenceListing 1.0
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cgtcgggtcg ggcggcttct 20
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taatacgact cactataggg agaggtgtga tggcgtccat aaa 43
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<400>3
ccaccgtcac cgtcttccac 20
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aatacgactc actataggga gaacctccgc cttgttcatg taaa 44
<210>5
<211>19
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<400>5
agaaacggct accacatcc 19
<210>6
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<400>6
taatacgact cactataggg agacaccaga cttgccctcc a 41
<210>7
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
ggccgcggcg ctcgcgcctt tttatctata gctggtgt 38
<210>8
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
tgtggggcgg cgtggacctt tttatctata gctggtgt 38
<210>9
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
acgccccggc ggggttcatt ttcgcagtgc tcgagctctg agc 43
<210>10
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
accccaccgt caccgtcttt ttcgcagtgc tcgagctctg agc 43
<210>11
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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ttcacgtgta tgacatcctt ttcgcagtgc tcgagctctg agc 43
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<212>DNA
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gcgtacagca tgcgcgccgc ttttctatgt atctgtgagt 40
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<213> Artificial Sequence (Artificial Sequence)
<400>13
ccagctccac gagcgattta ttttctatgt atctgtgagt 40
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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<210>15
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gggaccgtca tcacgcttct ttttcgcagt gctcgagctc tgagc 45
<210>16
<211>45
<212>DNA
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aaggaaggca gcaggctttt atctgtatag tgtctg 36
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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tcagatcact atgtactttt cgcagtgctc gagctctgag c 41
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
<400>25
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<210>26
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
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Claims (2)
1. A colloidal gold chromatography kit for jointly detecting herpes simplex virus I/II nucleic acid is characterized in that the kit is based on RNA isothermal amplification-gold probe chromatography technology and comprises the following components:
1) amplification reaction solution: containing 40mM Tris-HCl (pH 8.0), 12mM MgCl270mM KCl, 15% DMSO, 5mM DTT, 1mM of each dNTP, 2mM of each NTP, and 0.2 mu M of each amplification primer; wherein the amplification primers comprise three pairs: the gene comprises I type herpes simplex virus, II type herpes simplex virus and human reference genes, and specifically comprises the following genes:
(1) type I herpes simplex virus amplification primers:
HSVI-F（5’-3’）：CGTCGGGTCGGGCGGCTTCT；
HSVI-R（5’-3’）：TAATACGACTCACTATAGGGAGAGGTGTGATGGCGTCCATAAA；
(2) type II herpes simplex virus amplification primers:
HSVII-F（5’-3’）：CCACCGTCACCGTCTTCCAC；
HSVII-R（5’-3’）：AATACGACTCACTATAGGGAGAACCTCCGCCTTGTTCATGTAAA；
(3) amplification primers for reference genes:
18S-F（5’-3’）：AGAAACGGCTACCACATCC；
18S-R（5’-3’）：TAATACGACTCACTATAGGGAGACACCAGACTTGCCCTCCA；
2) amplification enzyme: comprises three types, reverse transcriptase, T7RNA polymerase and RnaseH; preferably the reverse transcriptase is AMV or M-MLV;
3) nucleic acid extraction reagent: cell lysate, purchased from Signosis, USA, Cat # CL-0001;
4) detection liquid: contain the nucleic acid gold probe of colloidal gold granule mark, the specific probe of every index, C line color development probe, every index specific probe has two kinds, is CES series and LES series respectively, and wherein CES series and LES series can design many again, specifically as follows:
(1) type I herpes simplex virus specific probe (5 '-3')
HSVI-CES1：GGCCGCGGCGCTCGCGCCTTTTTATCTATAGCTGGTGT；
HSVI-CES2：TGTGGGGCGGCGTGGACCTTTTTATCTATAGCTGGTGT；
HSVI-LES1：ACGCCCCGGCGGGGTTCATTTTCGCAGTGCTCGAGCTCTGAGC；
HSVI-LES2：ACCCCACCGTCACCGTCTTTTTCGCAGTGCTCGAGCTCTGAGC；
HSVI-LES3：TTCACGTGTATGACATCCTTTTCGCAGTGCTCGAGCTCTGAGC；
(2) Type II herpes simplex virus specific probe (5 '-3')
HSVII-CES1: GCGTACAGCATGCGCGCCGCTTTTCTATGTATCTGTGAGT；
HSVII-CES2: CCAGCTCCACGAGCGATTTATTTTCTATGTATCTGTGAGT；
HSVII-LES1: TGGACGCCATCACGCCCGCCTTTTCGCAGTGCTCGAGCTCTGAGC；
HSVII-LES2: GGGACCGTCATCACGCTTCTTTTTCGCAGTGCTCGAGCTCTGAGC；
HSVII-LES3: GGGTCTGACCCCCGAAGGCCTTTTCGCAGTGCTCGAGCTCTGAGC；
(3) Internal reference probe (5 '-3')
Internal reference (CES 1: AAGGAAGGCAGCAGGCttttATCTGTATAGTGTCTG);
internal reference (CES 2: GCGCAAATTACCCACTttttATCTGTATAGTGTCTG);
internal reference LES1: CCCGACCCGGGGAGGTttttCGCAGTGCTCGAGCTCTGAGC;
internal reference LES2: AGTGACGAAAAATAACttttCGCAGTGCTCGAGCTCTGAGC;
internal reference LES3: AATACAGGACTCTTTCttttCCGCAGTGCTCGAGCTCTGAGC;
(4) c line color probe (5 '-3')
TCAGATCACTATGTACttttCGCAGTGCTCGAGCTCTGAGC；
(5) Gold probe
The 5' end of the gold probe is modified by sulfhydrylation, and the sequence is as follows:
5’-CCTACTCTGCAGTGCTCCATCGTACGTCTGTCATTTTTGCTCAGAGCTCGAGCACTGCG-3’ ；
5) the test paper strip: the test strip is fixed on a PVC bottom plate, and a sample pad, an NC membrane and absorbent paper are arranged from left to right in sequence; the NC film is provided with a C line (quality control line) and three T lines (detection lines), and the directions from the sample pad to the absorbent paper are respectively HSVII-T, HSVI-T, reference-T and C lines (as shown in figure 3); an HSVI coated probe is coated at an HSVI-T position, an HSVII-T coated probe is coated at an HSVII-T position, an internal reference coated probe is coated at an internal reference-T position, and a C line coated probe is coated at a C line, wherein the specific sequence (5 '-3') is as follows:
c, coating of a probe: GTACATAGTGATCTGAttttGTACATAGTGATCTGA, respectively;
coating a probe with an internal reference line: CAGACACTATACAGATttttCAGACACTATACAGAT, respectively;
HSVI coil-coated probe: ACACCAGCTATAGATAttttACACCAGCTATAGATA, respectively;
HSVII coil-coated probes: ACTCACAGATACATAGttttACTCACAGATACATAG are provided.
2. Use of the kit of claim 1 for the preparation of a herpes simplex virus type I and/or type II detection reagent.
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| CN114790493A (en) * | 2021-11-04 | 2022-07-26 | 江汉大学 | MNP (protein-binding protein) marker site of herpes simplex virus, primer composition, kit and application of MNP marker site |
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