CN109487013B - Herpes simplex virus type I and type II detection marker, primer probe pair, kit and detection method - Google Patents
Herpes simplex virus type I and type II detection marker, primer probe pair, kit and detection method Download PDFInfo
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Abstract
The invention relates to the technical field of detection of pathogenic microorganisms, in particular to a herpes simplex virus I type and II type detection marker, a primer pair, a kit and a detection method. The invention takes the inverted repeat sequences RL2 and RS1 in the HSV genome as target regions, on one hand, 2 copies exist in the genome, the detection sensitivity can be further improved, and on the other hand, the nucleic acid similarity between the types is low, and the cross reaction is not easy to cause.
Description
Technical Field
The invention relates to the technical field of detection of pathogenic microorganisms, in particular to a herpes simplex virus I and/or II detection marker, a primer pair, a kit and a detection method.
Background
Herpes simplex is a viral skin disease caused by herpes simplex virus, and causes various diseases in humans, such as gingivitis, keratoconjunctivitis, encephalitis, reproductive system infection and neonatal infection.
Herpes Simplex Virus (HSV) belongs to the subfamily a virus of the family herpesviridae, and the size of the virus plasmid is about 180 nanometers. The viruses are currently classified into type I (HSV-1) and type II (HSV-2) according to the difference in antigenicity. HSV-1 is mainly obtained from the lesions of the lips and can also be separated from the lesions of the genitals, and HSV-2 can be separated from the lesions of the genitals. Infection is due to human-to-human contact and is the most vulnerable virus, but only a part of the clinical cases. The disease can be divided into: herpes labialis, herpetic keratitis, herpetic dermatitis, pudendal herpes, and Kaposi disease, and may also be causes of meningitis and encephalitis.
HSV can cause congenital diseases such as abortion, fetal deformity, mental retardation and the like due to placenta infection, and can cause infection of newborns when passing through HSV-2 infected birth canals, so that hyperpyrexia, dyspnea and central nervous system lesion occur. The necessary labor check and early anti-infective treatment can effectively reduce the spread of the virus to the next generation in HSV-2 patients.
The HSV genome is a linear DNA molecule and consists of a long fragment (L) and a short fragment (S) which are covalently connected, each fragment contains a single sequence and an inverted repeat sequence, wherein the single sequence of the long fragment is respectively UL1-UL56, the other 2 fragments are named as UL26.5 and UL49A, the inverted repeat sequence of the long fragment is respectively RL1 and RL2, the single sequence of the short fragment is respectively US1-US12, the other 1 fragment is named as US8A, and the inverted repeat sequence of the short fragment is RS 1. The genome encodes 70 different proteins, and 18 of them constitute viral DNA binding proteins and various enzymes, and are involved in viral DNA synthesis, packaging, nucleotide metabolism, etc., except for the characteristics of 24 proteins. More than 30 different proteins constitute virus structural proteins (such as capsid protein and envelope protein), and play an important role in protecting HSV DNA, causing HSV pathogenicity and inducing immune response of organisms.
At present, the detection method of herpes simplex virus mainly comprises the separation culture of bacteria, immunological diagnosis and molecular biological diagnosis, and the three methods have the following characteristics:
1) virus isolation and culture are reliable basis for clinically and clearly diagnosing herpes virus infection at present. The preparation method comprises the steps of collecting specimens such as vesicular fluid, cerebrospinal fluid, corneal scrapings, saliva and the like of pathological parts such as skin, genitals and the like, inoculating human diploid fibroblast strains WI38 and other subcultured cell strains such as Vero, BHK and the like, and after 24-48 hours, swelling, rounding, cell fusion and other pathological changes of cells occur. The virus isolation and culture time period is long, the operation is complicated, and professional operators and strict laboratory conditions are required, so that the application in clinical rapid diagnosis is few.
2) The immunological diagnosis also has hysteresis, cannot accurately reflect whether the current disease is infected or carries pathogens, and is difficult to meet the requirement of early diagnosis.
3) Pathogen nucleic acid detection: the kit has the advantages of rapidness, accuracy, short detection window, high sensitivity and the like, can make early diagnosis on pathogens, and provides powerful technical support for rapid analysis and control of epidemic situations.
The detection principle is that a detection probe is oligonucleotide comprising a 5 'end report group and a 3' end quenching group, when the probe is complete, the fluorescence emitted by the report group is greatly reduced because the quenching group is close to the report group, when a primer is extended, the probe combined with a template is cut off by Taq enzyme (5 '→ 3' exonuclease activity), and the report group is separated from the quenching group to generate a fluorescence signal. In each PCR cycle, a new reporter group is cut off, so that the increase of the fluorescence signal intensity is proportional to the quantity of the amplification product, and the amplification condition can be monitored in real time.
Most of the existing products adopt a single fluorescent quantitative PCR detection method aiming at HSV-1/HSV-2, and a clinical sample is required to be subjected to tube detection to draw conclusions respectively, so that the detection cost and the operation complexity are increased. A large number of reports are reported at home and abroad, HSV-1/HSV-2 nucleic acid typing detection is introduced, and the applied targets mainly comprise: glycoprotein G (US 4, nucleic acid similarity 78.4%), glycoprotein J (US 5, nucleic acid similarity 69.8%), glycoprotein D (US 6, nucleic acid similarity 79.2%), glycoprotein I (US 7, nucleic acid similarity 69.6%), glycoprotein E (US 8, nucleic acid similarity 67.3%), glycoprotein B (UL 27, nucleic acid similarity 77.3%), DNA polymerase (UL 30, nucleic acid similarity 89.1%; UL42, nucleic acid similarity 76.6%), thymidine kinase (UL 23, nucleic acid similarity 79.9%), and the like. The target genes are single copies, and missing detection is easily caused in the detection process due to insufficient abundance. And because the similarity of nucleic acids between two HSV types is over 65 percent, even the similarity of some genes is as high as 80 to 90 percent. The high similarity between HSV-1 and HSV-2 creates difficulties in typing the HSV.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a herpes simplex virus type I and/or type II detection marker, a primer pair, a kit and a detection method.
The invention provides a primer and a probe group for distinguishing HSV-1 virus and HSV-2 virus, which comprise
2 primers of nucleotide sequences shown in SEQ ID NO 1-2;
2 primers of nucleotide sequences shown in SEQ ID NO. 4-5;
a probe of a nucleotide sequence shown in SEQ ID NO. 3;
a probe of a nucleotide sequence shown as SEQ ID NO. 6.
The 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 3 is marked with a fluorescence reporter group CY5, and the 3' end is marked with a fluorescence quenching group BHQ 3;
the 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 6 is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1.
The primer and the probe are designed aiming at the RL2 fragment of HSV-1 and the RS1 fragment of HSV-2 respectively, have good accuracy, sensitivity and specificity, and can well distinguish the type I and the type II of HSV even if the detection is carried out in the same amplification system. The lowest detection limit can reach 5 copies.
The fluorescent reporter group of the probe is selected from FAM, JOE or HEX or VIC, ROX, CY3 or CY5, Texas Red; the fluorescence quenching group is selected from BHQ1, BHQ2, BHQ3, Dabcy1, MGB and TAMRA. In the embodiment of the invention, the fluorescent group of the HSV-1 virus detection probe is CY5, and the quenching group is BHQ 3. The fluorescent group of the HSV-2 virus detection probe is FAM, and the quenching group is BHQ 1.
The invention also provides a kit for detecting and distinguishing HSV-1 virus and HSV-2 virus, which comprises:
2 primers of nucleotide sequences shown in SEQ ID NO 1-2;
2 primers of nucleotide sequences shown in SEQ ID NO. 4-5;
a probe shown as SEQ ID NO. 3;
the probe shown in SEQ ID NO. 6.
The kit also comprises a fluorescent quantitative PCR detection reagent and/or a sample extraction reagent;
the fluorescent quantitative PCR detection reagent comprises qPCR Master Mix enzyme mixed liquor and qPCR Master Mix reaction buffer solution.
The sample extraction reagent comprises NaOH, TritonX-100 and TE buffer solution.
The qPCR Master Mix enzyme mixed solution comprises Taq enzyme and UNG enzyme, wherein the Taq enzyme is AntartTaq DNA polymerase, and the UNG enzyme is uracil-N-glycosylase.
The sample extracting solution comprises NaOH with the final concentration of 0.01% -0.5%, and preferably 0.1%.
The sample extracting solution comprises TritonX-100 with the final concentration of 0.01-0.5%, preferably 0.1%.
Further, the kit also comprises a positive control and/or a negative control.
The negative control of the present invention was DEPC water.
The positive reference substance is a plasmid solution containing HSV-1 target gene fragments. The HSV-1 specific target gene fragment sequence is as follows: GGTGCTGATTGACGCGGGAAATCCCCCCC CATTCTTACCCGCCCCCCTTTTTTCCCCTTAGCCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGACCTGCCGGACAGCAG (SEQ ID NO. 10). Preferably, the concentration of the plasmid solution containing the HSV-1 target gene fragment is 10000 copies/mu L, and the corresponding Ct value is about 23.
The positive reference substance is a plasmid solution containing HSV-2 target gene fragments. The HSV-2 specific target gene fragment sequence is as follows: GTCGTCGTCGTCGTCGTCGTCAGACGAGGAG GCGGATGCAGACGAGGAGGAGGAGGCGGAGGAGGAGGCGGAGGACGCCGACGACGAGGATCCGGATTTTGATGAGTCAGAGGCGGCCGAG (SEQ ID NO. 11). Preferably, the concentration of the plasmid solution containing the HSV-2 target gene fragment is 10000 copies/mu L, and the corresponding Ct value is about 23.
Further, the kit also comprises an internal reference primer and a probe. The internal reference gene adopted by the invention is HBB, and the sequence of the HBB specific target gene fragment is as follows: TCTGACTCCTGAGG AGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATG (SEQ ID No. 12). Primers for HBB included: 2 primers of nucleotide sequences shown in SEQ ID NO 7-8. The nucleotide sequence of the probe for HBB is shown in SEQ ID NO. 9. The fluorescent group of the HBB-P probe is ROX, and the quenching group is BHQ 1.
The invention also provides a method for detecting or distinguishing HSV-1 virus from HSV-2 virus, which comprises the following steps: the kit for detecting or distinguishing HSV-1 virus and HSV-2 virus carries out real-time fluorescent quantitative PCR detection on the DNA of a sample to be detected, and the generated S-type amplification curve is positive.
The real-time fluorescent quantitative PCR system comprises:
qPCR Master Mix enzyme mixture 1. mu.L;
qPCR Master Mix reaction buffer 5. mu.L;
1 mu L of each primer;
0.4. mu.L of each probe;
make up to 25. mu.L with water.
In the system, the concentration of the primer is 100-1000 nM, preferably 500 nM. The concentration of the probe is 10-500 nM, preferably 200 nM.
The reaction buffer is qPCR Master Mix reaction buffer, and in some embodiments, Antart qPCR Master Mix reaction buffer is used.
The enzyme mixed solution is qPCR Master Mix enzyme mixed solution. In some embodiments, an Anstart qPCR Master Mix enzyme cocktail is used.
The real-time fluorescent quantitative PCR program comprises:
2 minutes at 50 ℃;
pre-denaturation at 95 ℃ for 5 min;
95 ℃ for 15 seconds → 60 ℃ for 35 seconds, 45 cycles.
The invention also provides a method for detecting or distinguishing HSV-1 virus from HSV-2 virus, comprising: the kit for detecting or distinguishing HSV-1 virus and HSV-2 virus carries out real-time fluorescent quantitative PCR detection on the DNA of a sample to be detected, and the generated S-type amplification curve is positive.
The real-time fluorescent quantitative PCR system comprises:
1 mu L of qPCR Master Mix enzyme mixed solution;
qPCR Master Mix reaction buffer 5. mu.L;
1 μ L of each primer;
make up to 25. mu.L with water.
In the system, the concentration of the primer is 100-1000 nM, preferably 500 nM. The reaction buffer contains dye (such as SybrGreen, EvaGreen) qPCR Master Mix reaction buffer, and in some embodiments, dye-containing Anstart qPCR Master Mix reaction buffer is used.
The enzyme mixed solution is qPCR Master Mix enzyme mixed solution. In some embodiments, an Anstart qPCR Master Mix enzyme cocktail is used.
The real-time fluorescent quantitative PCR program comprises:
2 minutes at 50 ℃;
pre-denaturation at 95 ℃ for 5 min;
95 ℃ for 15 seconds → 60 ℃ for 35 seconds, 45 cycles.
The judgment of the result specifically includes:
the Ct value of the amplification curve of the DNA of the sample to be detected is less than 38, and the amplification curve has a typical S-shaped amplification curve and is a positive result;
the Ct value of the amplification curve of the DNA of the sample to be detected is equal to 45 (or shown as Undet) or no typical S-shaped amplification curve, and the result is negative;
the Ct value of the amplification curve 38 of the DNA sample to be detected is not more than 45, the DNA sample is a gray area, if the retest result is still Ct value less than 45 and a typical S-shaped amplification curve exists, the DNA sample is judged to be a positive result; if the Ct value is not displayed or no typical S-type amplification curve exists, judging as a negative result;
and under the condition that the HSV-1 and HSV-2 samples to be detected are negative, the amplification condition of HBB is inspected, or HBB has no S-type amplification curve, or the Ct value is more than 35, which indicates that sampling failure, extraction failure or sample adding error needs to be retested.
For experiments where positive controls were set, it should also be satisfied that: positive control, CY5, FAM channel and ROX channel all have obvious amplification, have typical S-type amplification curve, and Ct value is less than 30;
for the experiments where negative controls were set, it should also be satisfied: negative controls, CY5, FAM channel, ROX channel Ct value =45 (or shown as undet) or no canonical sigmoid amplification curve, for subsequent determination, otherwise a question-finding should be performed.
The primer and the probe can be used for detecting samples from various sources. Detection of a biological sample can determine whether the sample is infected with HSV-1 or HSV-2, while detection of a non-biological sample can determine whether the sample is contaminated with HSV-1 or HSV-2. Thus, the kits of the present invention may be used for the detection of a sample for non-diagnostic purposes. In the present invention, the sample to be tested is derived from herpes, vagina, urinary tract, medical device, drug, food or cosmetic. In some embodiments, the sample to be tested is vaginal secretion, urinary tract secretion and/or herpes bleb fluid.
The beneficial effects of the invention at least comprise:
(1) the invention takes the inverted repeat sequences RL2 and RS1 in the HSV genome as target regions, on one hand, 2 copies exist in the genome, the detection sensitivity can be further improved, and on the other hand, the nucleic acid similarity between the types is low, so that the cross reaction is not easy to cause;
(2) the invention designs 6 primers and 3 Taqman probes aiming at conserved regions of HSV-1/HSV-2/HBB, has high specificity by combining with the conserved regions of target genes, and has high specificity by combining with a pseudomonas aeruginosa strain CMCC10104, a staphylococcus aureus strain CMCC26001, a neisseria meningitidis strain CMCC29108, a neisseria meningitidis strain CMCC29204, a lactobacillus casei strain CMCC34103, an escherichia coli strain CMCC44103, a proteus strain CMCC49102, a salmonella typhi strain CMCC50096, a shigella flexneri strain CMCC51573, a candida albicans strain CMCC98001, a Mycoplasma Hominis (MH), a toxoplasma Gondii (GD), a papillomavirus (human milk), a Human Cytomegalovirus (HCMV), a Mycoplasma Pneumoniae (MP), a Mycoplasma Genitalium (MG), a bacillus Gardneri (GV), a candida albicans (CC), a trichomonas vaginalis (Tv), a gonococcus (NG), a trachoma (CT), a Chlamydia Trachomatis (CT), The Ureaplasma Urealyticum (UU) and the Ureaplasma Parvum (UP) have no cross reaction;
(3) HSV-1/HSV-2 typing detection is completed in the same tube and is distinguished by different fluorescence labeled probes, typing detection of 2 pathogens can be completed clinically through one-time sample nucleic acid extraction and one-time nucleic acid sample addition, the problem of overhigh detection cost caused by previous single-tube detection is effectively reduced, and the complicated operation condition of single-tube detection is effectively improved;
(4) target gene fragments are shorter and are all between 100 and 150bp, if the target gene fragments are too short, the decontamination effect of UNG enzyme is influenced, and if the target gene fragments are too long, the reaction efficiency is reduced, so that the method can effectively avoid pollution, can ensure the high efficiency and sensitivity of the reaction, and can detect HSV-1/HSV-2 DNA with 5 copies;
(5) human beta-globin gene specific primer probes are added into the reaction tube, so that the whole process of sample sampling, sample extraction and PCR detection can be effectively monitored, and the defect that whether the sample sampling is successful or not can not be effectively monitored by adding external standards in the extraction process is overcome.
The kit is very suitable for clinical use and popularization, and has important significance for the prevention and control work of HSV-1/HSV-2 in China.
Drawings
FIG. 1 shows the fluorescence detection results of the specificity experiment of example 4, where the template for each detection curve is 1: pseudomonas aeruginosa strain CMCC10104, 2: staphylococcus aureus strain CMCC26001, 3: neisseria meningitidis strain CMCC29108, 4: lactobacillus casei strain CMCC34103, 5: escherichia coli strain CMCC44103, 6: proteus strain CMCC49102, 7: salmonella typhi strain CMCC50096, 8: shigella flexneri strain CMCC51573, 9: candida albicans strain CMCC98001, 10: mycoplasma Hominis (MH), 11: toxoplasma Gondii (GD), 12: human Papilloma Virus (HPV), 13: human Cytomegalovirus (HCMV), 14: mycoplasma Pneumoniae (MP), 15: mycoplasma Genitalium (MG), 16: gardnerella (GV), 17: candida albicans (CC), 18: trichomonas vaginalis (Tv), 19: gonococcus (NG), 20: chlamydia Trachomatis (CT), 21: ureaplasma Urealyticum (UU), 22: ureaplasma Parvum (UP);
FIG. 2 shows the fluorescence detection results of HSV-1 (Cy 5 channel) in the sensitivity experiment of example 7, the templates corresponding to the detection curves are 1-7 plasmid solutions containing HSV-1/HSV-2/HBB target gene fragments, and the concentrations are 1.0 × 10 in sequence 6 、1.0×10 5 、1.0×10 4 、1.0×10 3 、1.0×10 2 、1.0×10 1 、1.0×10 0 Copy/. mu.L;
FIG. 3 shows the fluorescence detection results of HSV-2 (FAM channel) in the sensitivity experiment of example 7, wherein the template corresponding to each detection curve is 1-7 plasmid solutions containing HSV-1/HSV-2/HBB target gene fragments, and the concentrations are 1.0 × 10 in sequence 6 、1.0×10 5 、1.0×10 4 、1.0×10 3 、1.0×10 2 、1.0×10 1 、1.0×10 0 Copy/. mu.L;
FIG. 4 shows the result of fluorescence detection of HSV-1 (Cy 5 channel) in 100-fold dilutions of the positive control (R1) in the replicate test of example 8;
FIG. 5 shows the results of fluorescence detection of HSV-2 (FAM channel) in 100-fold dilutions of the positive control (R1) from the replicate test of example 8.
Detailed Description
The invention provides a herpes simplex virus I and/or II detection marker, a primer-probe pair, a kit and a detection method. The skilled person can use the contents to modify the process parameters appropriately. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1: design of primer probe pair for rapidly detecting HSV-1/HSV-2
Aiming at HSV-1 RL2 gene, HSV2 RS1 gene and HBB gene sequences in NCBI, a primer pair and a probe are designed, and the sequences are as follows:
example 2: real-time fluorescent quantitative PCR (polymerase chain reaction) kit for rapidly detecting HSV-1
A real-time fluorescent quantitative PCR kit for rapidly detecting human HSV-1 comprises an HSV-1 primer, an HSV-1 probe, an HBB primer, an HBB probe, enzyme mixed liquor, reaction buffer solution, sample extracting solution, a positive reference substance, a negative reference substance, an instruction book and a kit body.
Wherein the primer HSV-1-F, HSV-1-R sequence is shown as SEQ ID NO.1-2 in sequence, and the concentration is 500 nM; the sequence of the probe HSV1-P is shown in SEQ ID NO. 3, the fluorescent group is Cy5, the quenching group is BHQ3, and the concentration is 200 nM.
Wherein the sequence of the primer HBB-F, HBB-R is shown as SEQ ID NO.7-8 in sequence, and the concentration is 500 nM; the sequence of the probe HBB-P is shown in SEQ ID NO. 9, the fluorescent group of the probe is ROX, the quenching group is BHQ1, and the concentration is 200 nM.
The Antart qPCR Master Mix enzyme mixture and the Antart qPCR Master Mix5 × reaction buffer were supplied from Fenpeng Bio Inc., the Antart qPCR Master Mix was diluted 25-fold and the Antart qPCR Master Mix5 × reaction buffer was diluted 5-fold.
Wherein the sample extract is TE buffer solution containing 0.1% NaOH and 0.1% TritonX-100.
The positive control substance is a plasmid solution containing HSV-1 and HBB target gene fragments, the concentration is 10000 copies/mu L, and the corresponding Ct values are all about 23.
Wherein the negative control is DEPC water.
Wherein the positive control comprises: the sequence of the HSV-1 specific target gene fragment is as follows:
GGTGCTGATTGACGCGGGAAATCCCCCCCCATTCTTACCCGCCCCCCTTTTTTCCCCTTAGCCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGACCTGCCGGACAGCAG（SEQ ID No.10）。
wherein the positive control comprises: the sequence of HBB specific target gene fragment is:
TCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATG（SEQ ID No.12）。
example 3: real-time fluorescent quantitative PCR (polymerase chain reaction) kit for rapidly detecting HSV-2
A real-time fluorescent quantitative PCR kit for rapidly detecting human HSV-2 comprises an HSV-2 primer, an HSV-2 probe, an HBB primer, an HBB probe, enzyme mixed liquor, reaction buffer solution, sample extracting solution, a positive reference substance, a negative reference substance, an instruction book and a kit body.
Wherein the primer HSV-2-F, HSV-2-R sequence is shown as SEQ ID NO.4-5 in sequence, and the concentration is 500 nM; the sequence of the probe HSV-2-P is shown as SEQ ID NO. 6, the fluorescent group is FOM, the quenching group is BHQ1, and the concentration is 200 nM.
Wherein the primer HBB-F, HBB-R sequence is shown as SEQ ID NO.7-8 in sequence, and the concentration is 500 nM; the sequence of the probe HBB-P is shown in SEQ ID NO. 9, the fluorescent group of the probe is ROX, the quenching group is BHQ1, and the concentration is 200 nM.
The Antart qPCR Master Mix enzyme mixture and the Antart qPCR Master Mix5 × reaction buffer were supplied from Fenpeng Bio Inc., the Antart qPCR Master Mix was diluted 25-fold and the Antart qPCR Master Mix5 × reaction buffer was diluted 5-fold.
Wherein the sample extract is TE buffer solution containing 0.1% NaOH and 0.1% TritonX-100.
The positive control substance is a plasmid solution containing HSV-1 and HBB target gene fragments, the concentration is 10000 copies/mu L, and the corresponding Ct values are all about 23.
Wherein the negative control is DEPC water.
Wherein the positive control comprises: the HSV-2 specific target gene fragment sequence is as follows:
GTCGTCGTCGTCGTCGTCGTCAGACGAGGAGGCGGATGCAGACGAGGAGGAGGAGGCGGAGGAGGAGGCGGAGGACGCCGACGACGAGGATCCGGATTTTGATGAGTCAGAGGCGGCCGAG（SEQ ID No.11）。
wherein the positive control comprises: the sequence of the HBB specific target gene fragment is as follows:
TCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATG（SEQ ID No.12）。
example 4: real-time fluorescent quantitative PCR (polymerase chain reaction) kit for rapidly detecting and distinguishing HSV-1/HSV-2
The real-time fluorescent quantitative PCR kit for rapidly detecting and distinguishing HSV-1/HSV-2 comprises an HSV-1 primer, an HSV-1 probe, an HSV-2 primer, an HSV-2 probe, an HBB primer, an HBB probe, enzyme mixed liquor, reaction buffer solution, sample extracting solution, a positive control, a negative control, an instruction and a kit body.
Wherein the primer HSV-1-F, HSV-1-R sequence is shown as SEQ ID NO.1-2 in sequence, and the concentration is 500 nM; the sequence of the probe HSV1-P is shown in SEQ ID NO. 3, the fluorescent group is Cy5, the quenching group is BHQ3, and the concentration is 200 nM.
Wherein the primer HSV-2-F, HSV-2-R sequence is shown as SEQ ID NO.4-5 in sequence, and the concentration is 500 nM; the sequence of the probe HSV-2-P is shown in SEQ ID NO. 6, the fluorescent group is FOM, the quenching group is BHQ1, and the concentration is 200 nM.
Wherein the primer HBB-F, HBB-R sequence is shown as SEQ ID NO.7-8 in sequence, and the concentration is 500 nM; the sequence of the probe HBB-P is shown as SEQ ID NO. 9, the fluorescent group of the probe is ROX, the quenching group is BHQ1, and the concentration is 200 nM.
The Antart qPCR Master Mix enzyme mixture, Antart qPCR Master Mix5 Xreaction buffer were supplied from Fipeng Bio Inc., the Antart qPCR Master Mix was used diluted 25-fold, and the Antart qPCR Master Mix5 Xreaction buffer was used diluted 5-fold.
Wherein the sample extract is TE buffer solution containing 0.1% NaOH and 0.1% TritonX-100.
The positive control substance is a plasmid solution containing HSV-1/HSV-2/HBB target gene fragments, the concentration is 10000 copies/mu L, and the corresponding Ct values are all around 23.
Wherein the HSV-1 specific target gene fragment has the sequence:
GGTGCTGATTGACGCGGGAAATCCCCCCCCATTCTTACCCGCCCCCCTTTTTTCCCCTTAGCCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGACCTGCCGGACAGCAG（SEQ ID No.10）。
wherein the HSV-2 specific target gene fragment sequence is as follows:
GTCGTCGTCGTCGTCGTCGTCAGACGAGGAGGCGGATGCAGACGAGGAGGAGGAGGCGGAGGAGGAGGCGGAGGACGCCGACGACGAGGATCCGGATTTTGATGAGTCAGAGGCGGCCGAG（SEQ ID No.11）。
wherein the sequence of the HBB specific target gene fragment is as follows:
TCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATG（SEQ ID No.12）。
wherein the negative control is DEPC water.
Example 5: rapid detection method of HSV-1/HSV-2 nucleic acid detection kit
The kit of embodiment 4 is used for rapidly detecting HSV-1/HSV-2 in human vaginal secretion, urinary tract secretion and vesicle fluid samples, and comprises the following specific steps:
(1) nucleic acid extraction: adding 1mL of physiological saline into a collecting pipe of 4 cases (sample numbers 1-4) of vaginal secretion, 3 cases (sample numbers 5-7) of urinary tract secretion and 3 cases (sample numbers 8-10) of bleb liquid sample (adopting a virus isolation culture and typing method to determine whether the bleb liquid is infected by HSV-1 or HSV-2, wherein the method is the current industrial gold standard), fully oscillating and washing a cotton swab, then squeezing and discarding the cotton swab by the wall, transferring 500 mu L of liquid into a 1.5mL centrifuge tube, centrifuging at 13000rpm for 5 minutes, discarding supernatant, adding 1mL of physiological saline into the precipitate, scattering the precipitate, centrifuging at 13000rpm for 5 minutes, discarding supernatant, adding 50 mu L of sample extracting solution which is oscillated and mixed into the precipitate, oscillating and scattering the precipitate on a vortex oscillator (if necessary, scattering the precipitate by using a gun head), carrying out dry bath at 100 ℃ or water bath for 10 minutes, centrifuging at 13000rpm for 5 minutes, the supernatant was used for PCR reaction (the pellet at the bottom of the tube was not touched during aspiration).
(2) Real-time quantitative fluorescent PCR: and carrying out PCR amplification reaction by using the HSV-1/HSV-2 real-time fluorescent quantitative PCR detection kit, and respectively taking the extracted DNA of the sample to be detected, and 5 muL of each of the positive control and the negative control as templates.
Respectively using an HSV-1 primer and a probe; HSV-2 primers and probes; HSV-1 and HSV-2 primers and probes are used for detection.
Mixing the template with the reagent of the kit, and carrying out real-time fluorescent quantitative PCR reaction, wherein the PCR reaction conditions are as follows: UNG enzyme decontamination at 50 ℃ for 2 minutes, pre-denaturation at 95 ℃ for 5 minutes, 15 seconds → 60 ℃ for 35 seconds, Cy5, FAM, ROX channels for collection of fluorescence signals for detection, for 45 cycles.
(3) And (5) judging a result:
the following requirements are met: positive control, FAM channel, CY5 channel and ROX channel all have obvious amplification, have typical S-type amplification curve, and Ct value is less than 30; negative control, FAM channel, CY5 channel, ROX channel Ct value =45 (or shown as undet) or no typical S-type amplification curve, and performing subsequent judgment, otherwise, performing problem search;
the Ct value of an amplification curve of the DNA of the sample to be detected is less than 38, and the amplification curve has a typical S-shaped amplification curve and is a positive result;
the Ct value of the amplification curve of the DNA of the sample to be detected is equal to 45 (or shown as Undet) or no typical S-shaped amplification curve, and the result is negative;
fourthly, if the Ct value of the amplification curve 38 of the DNA sample to be detected is not more than 45 and is a gray area, if the retest result is still the Ct value less than 45 and has a typical S-shaped amplification curve, the result is judged to be a positive result; if the Ct value is not displayed or no typical S-type amplification curve exists, judging that the result is negative;
The results are shown in Table 2:
the results show that 3 parts of HSV-1 positive samples, 3 parts of HSV-2 positive samples, 1 part of double positive samples and 3 parts of negative samples. The result is consistent with the identification in the prior art, and the accuracy can reach 100 percent.
Example 6: experiment of specificity
The kit of example 4 and the method of example 5 were used to perform PCR detection on Pseudomonas aeruginosa strain CMCC10104, Staphylococcus aureus strain CMCC26001, Neisseria meningitidis strain CMCC29108, Neisseria meningitidis strain CMCC29204, Lactobacillus casei strain CMCC34103, Escherichia coli strain CMCC44103, Proteus mutans strain CMCC49102, Salmonella typhi strain CMCC50096, Shigella flexneri strain CMCC51573, Candida albicans strain CMCC98001, Mycoplasma Hominis (MH), Toxoplasma Gondii (GD), Human Papilloma Virus (HPV), Human Cytomegalovirus (HCMV), Mycoplasma Pneumoniae (MP), Mycoplasma Genitalium (MG), Gardnerella (MG), Candida albicans (CC), Trichomonas vaginalis (Tv), gonococcus (CT), Chlamydia urealyticus (UU), and Urea Parvum (UP), and the results are shown in FIG. 1, respectively, and the results show that all samples are negative. The result shows that the detection kit has high specificity and has no cross reaction with common vagina and reproductive tract infection samples.
Example 7: sensitivity test
Taking a positive control (namely plasmid solution containing HSV-1/HSV-2/HBB target gene fragments), determining the concentration of the positive control, calculating the copy number, diluting according to a 10-fold concentration gradient, selecting 1.0 multiplied by 10 0 ～1.0×10 6 Copies/. mu.L (corresponding to a viral concentration of 50CFU/mL to 5X 10 7 CFU/mL) as a sample, using the kit and the detection method of the present invention.
The detection results are shown in figures 2-3, and the results show that the kit has the lowest detection limit concentration of 1.0 multiplied by 10 for HSV-1/HSV-2 0 At a concentration of 50CFU/mL, i.e., at a minimum detection limit of 5 copies (corresponding to a concentration of 50 CFU/mL).
Example 8: repeatability test
Taking 1000 times of the dilution of the positive control in the kit, named as R1, using the kit and the detection method of the invention, repeating the test continuously for 10 times, the detection result is shown in figure 4-figure 5, table 3, the result shows that the Ct value variation coefficient of 100 times of the dilution of the positive control is less than 5%.
Example 9: influence of gynecological drugs on sample detection
Common gynecological drugs and cleaning products on the market are selected, including a chylomicron suppository, a nifuratel nystatin vaginal soft capsule, a meifukang carbomer gel, a cervicitis-cleaning carbomer gel, an ofloxacin gel, a metronidazole furanone suppository and a jieeryin lotion, and are tested to verify the influence of the suppository on the detection of the kit.
The results show that 0.1mg/mL of the Xiaomi suppository, 10mg/mL of the nifuratel nysfungin vaginal soft capsule, 10mg/mL of the Meifukang carbomer gel, 10mg/mL of the cervicitis cleaning carbomer gel, 10mg/mL of the ofloxacin gel, 10mg/mL of the metronidazole furanone suppository and 0.1% of the Jieeryin lotion have no interference on the detection. The kit provided by the invention can realize whether HSV-1 or HSV-2 infection exists in the medicine.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
<110> Zhongsheng Fangzheng Biotechnology Ltd
<120> herpes simplex virus type I and type II detection marker, primer probe pair, kit and detection method
<130> MP1829941
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Claims (1)
1. The application of the primer and the probe group in the preparation of a kit for detecting and distinguishing HSV-1 virus and HSV-2 virus is characterized in that the primer and the probe group comprise:
2 primers of nucleotide sequences shown in SEQ ID NO 1-2;
2 primers of nucleotide sequences shown in SEQ ID NO. 4-5;
a probe of the nucleotide sequence shown in SEQ ID NO. 3; 3, the 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 3 is marked with a fluorescence reporter group CY5, and the 3' end is marked with a fluorescence quenching group BHQ 3;
a probe of the nucleotide sequence shown in SEQ ID NO. 6; the 5 'end of the probe of the nucleotide sequence shown in SEQ ID NO. 6 is marked with a fluorescence reporter FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1.
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| CN110343775A (en) * | 2019-07-12 | 2019-10-18 | 中生方政生物技术股份有限公司 | The primer of double check Gardner bacillus and trichomonas vaginalis, probe groups, kit and detection method |
| CN110343780A (en) * | 2019-07-12 | 2019-10-18 | 中生方政生物技术股份有限公司 | Gardner bacillus, the primer of Candida albicans and trichomonas vaginalis Multiple detection, probe groups, kit and detection method |
| CN110283921A (en) * | 2019-07-12 | 2019-09-27 | 中生方政生物技术股份有限公司 | Gardner bacillus, the primer of Candida albicans double check, probe groups, kit and detection method |
| CN112063749A (en) * | 2019-10-28 | 2020-12-11 | 北京康美天鸿生物科技有限公司 | Primer probe combination for identifying herpes simplex virus and fluorescent quantitative PCR kit |
| CN110904277B (en) * | 2019-12-25 | 2023-04-18 | 圣湘生物科技股份有限公司 | Compositions, kits and methods for HSV-1 and HSV-2 typing detection |
| CN111172323A (en) * | 2020-01-17 | 2020-05-19 | 郑州安图生物工程股份有限公司 | Herpes simplex virus 1 type and 2 type typing nucleic acid detection kit |
| CN113373267B (en) * | 2021-07-16 | 2022-04-29 | 浙江大学 | Multiplex fluorescence quantitative RT-PCR kit for detecting blood-borne infectious viruses |
| CN114790493B (en) * | 2021-11-04 | 2023-06-16 | 江汉大学 | MNP (MNP) marking site of herpes simplex virus, primer composition, kit and application of MNP marking site |
| CN113846190B (en) * | 2021-11-12 | 2023-02-17 | 河南省人民医院 | Triple fluorescent quantitative PCR (polymerase chain reaction) detection composition, method and kit for herpes simplex virus 1, herpes simplex virus 2 and pseudorabies virus |
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