CN109988853B - Primer and probe combination for chlamydia psittaci genotype detection and application - Google Patents
Primer and probe combination for chlamydia psittaci genotype detection and application Download PDFInfo
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Abstract
The invention provides a primer and probe combination for detecting Chlamydia psittaci genotype and application thereof. The C.psittaci genotype specific probe and primer are designed by analyzing and screening gene sequences of ompA genes VD1-VD 4 of Chlamydia psittaci according to the correspondence and equivalence between Chlamydia psittaci serotypes and ompA genotypes. The invention also provides a genotype identification chip of the ompA gene specific region prepared based on the probe. The invention provides a novel microarray chip method for quickly, effectively and high-flux detecting C.psittaci genotype, which has good sensitivity and specificity, provides a novel practical and effective detection method for diagnosing, analyzing and researching the genotype of Chlamydia psittaci, and provides an effective means for diagnosing, epidemiological investigation and infectious disease prevention and control of Chlamydia psittaci.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a primer and probe combination for chlamydia psittaci genotype detection and application thereof.
Background
Psittaci is a zoonosis pathogen parasitized in strict eukaryotic cells, is first isolated from psittaci, is a natural epidemic pathogen, has an extremely wide host range, and can cause infection of human, poultry, wild poultry and livestock and cause various diseases. The transmission of psittaci mainly occurs in birds and poultry, which causes serious respiratory diseases, digestive tract diseases and reduction of production performance of the birds, and the birds have long-term harm and loss due to recessive infection in a partial infection state. Psittaci, when a human is brought into contact with an animal infected with c, infectious aerosols, which are discharged from the respiratory tract of sick birds, secretions, diseased animals, or pathogens invade the human body through the conjunctiva and mucous membranes of the eyes, causing respiratory infections in humans, manifested as atypical pneumonia, and bacteremia, which are clinically known as psittacosis or ornithosis. Since the 50 s of the 20 th century, several outbreaks of psittacosis have been reported around the world in the population of poultry and turkeys. Nowadays, with the large-scale intensive breeding of poultry by human beings, poultry such as chickens, ducks, pigeons and turkeys become important infectious sources of c. psittaci infected by human beings, and the threat to the health of breeding personnel is more prominent under the environment polluted by high-concentration pathogens in intensive farms. Psittaci also infects a variety of mammals and domestic animals, resulting in pneumonia, enteritis, urogenital inflammation in animals such as cattle, sheep, pigs, and abortion, premature birth, weak or dead fetus in pregnant animals.
The invasiveness and pathogenicity of the psittaci to different hosts are different, the psittaci mainly depends on different serotypes of the psittaci, the C.psittaci is divided into 9 serotypes including A, B, C, D, E, E/B, F, WC and M56 at present, each serotype has certain host specificity, and the main hosts of the type A are parrots, pigeons and humans; the B type main host is pigeon, turkey, and cattle; the C-type main host is duck, goose, turkey, pine chicken and human; the main D-type hosts are turkeys, parrots and pigeons, and have strong infectivity on human beings; the host range of type E is the broadest, including pigeons, ducks, turkeys (low toxicity), and type E has also been isolated from human pneumonia cases; type F is very specific for parrots; type E/B is a newly identified serotype in recent years, duck being the main infectious host; WC type and M56 type are mainly spread among wild birds and livestock. The A type, the C type, the D type and the E type of the C.psittaci can be transmitted among people and cause infection and morbidity of people, and as pathogens of zoonosis infectious diseases, the A type, the C type, the D type and the E type of the C.psittaci not only cause huge threats and losses to the animal husbandry production, but also seriously threaten the health of human beings. Therefore, the detection of the serotype of psittaci has important significance for the detection of the pathogen, the monitoring of epidemic conditions and the prevention and control of human public health.
The current diagnostic methods for typing C.psittaci mainly include (1) typing C.psittaci serotypes using serotype-specific monoclonal antibodies, which lacks existing commercial sets of serotype-specific monoclonal antibodies, requires cell culture and professional purification of chlamydia strains to be tested, requires professional chlamydia culture techniques and corresponding culture conditions, and is limited in diagnosis for atypical isolates, (2) PCR-RF L P, performs restriction fragment length polymorphism analysis for ompA genes, which has limited sensitivity in clinical samples, and still requires cell culture of samples if better sensitivity is achieved.
At present, no mature kit for diagnosing the genotype of the chlamydia psittaci exists in the domestic market no matter based on a PCR-RF L P method or a fluorescent quantitative PCR method, meanwhile, the research on the genotype diagnosis of the zoonosis pathogen C.psittaci is less, and a maturation scheme for detecting and analyzing each genotype of C.psittaci with high flux and high efficiency by using a gene chip method is not available so far.
Disclosure of Invention
The invention aims to provide a primer and probe combination for detecting Chlamydia psittaci genotype and application thereof.
Another object of the present invention is to provide a gene chip for identification and diagnosis based on the genotype (serotype) of the specific region of ompA gene.
The invention has the following conception: according to the sequence analysis result of the c.psittaci gene, the nucleotide differences among the serotypes mainly occur in 4 variation regions of the ompA gene (VD1-VD 4). Researches find that the chlamydia psittaci serotype and the ompA genotype have correspondence and equivalence, and the ompA gene is used as a precise target gene for serotype typing of C.psittaci based on the biological information characteristics.
In order to realize the purpose of the invention, the C.psittaci genotype specific probe and specific primer are designed and obtained by analyzing and screening gene sequences of ompA genes VD1-VD 4 of Chlamydia psittaci according to the correspondence and equivalence between the serotype of Chlamydia psittaci (C.psittaci) and the ompA genotype thereof. The specific primer is modified by 5' Biotin, streptavidin-horseradish peroxidase (SA-HRP) is introduced into chip hybridization reaction, and a precipitable TMB substrate is added in the color development process, so that a precipitable color development spot is formed under the catalysis of HRP enzyme, the visual detection of C.psittaci is achieved, the genotype of the chip is identified, and the high-throughput detection and the visual identification of different genotypes of Chlamydia psittaci are realized.
In a first aspect, the invention provides primers for detecting Chlamydia psittaci genotype, comprising 2 pairs of primers, a primer pair 1 and a primer pair 2, the sequences of which are respectively shown as SEQ ID NO. 1-2 and SEQ ID NO. 3-4. 3, N represents a hypoxanthine base I; w represents A or T; m represents A or C, Y represents C or T, and R represents A or G.
In a second aspect, the present invention provides a probe set for the detection of chlamydia psittaci genotype, the probe set comprising:
chlamydia psittaci A genotype specific probe with sequence shown in SEQ ID NO 5-6;
a Chlamydia psittaci genotype B specific probe, the sequence of which is shown in SEQ ID NO. 7;
the Chlamydia psittaci C genotype specific probe has the sequence shown in SEQ ID NO 8-9;
chlamydia psittaci D genotype specific probe with sequence shown in SEQ ID NO 10-11;
chlamydia psittaci E genotype specific probe with sequence shown in SEQ ID NO 12-13;
chlamydia psittaci E/B genotype specific probe, the sequence is shown in SEQ ID NO. 14;
chlamydia psittaci F genotype specific probe with sequence shown in SEQ ID NO 15-17;
the Chlamydia psittaci WC genotype specific probe has the sequence shown in SEQ ID NO 18-19;
chlamydia psittaci M56 genotype specific probe, the sequence is shown in SEQ ID NO: 20.
In a third aspect, the invention provides a primer and probe combination for detecting Chlamydia psittaci genotype, which comprises the primer shown in SEQ ID NO. 1-4 and the probe combination shown in SEQ ID NO. 5-20.
In a fourth aspect, the present invention provides a chip for genotyping Chlamydia psittaci, i.e., a genotyping diagnostic gene chip based on the ompA gene specific region (serotype).
The chip comprises a chip carrier and a specific probe shown in SEQ ID NO. 5-20.
Preferably, the chip also comprises 2 positive control probes, and the sequences are respectively shown in SEQ ID NO 21-22; 1 quality control probe with the sequence shown in SEQ ID NO. 23; and a control probe which is a spotting diluent. 22, N represents a hypoxanthine base I; w represents A or T; m represents A or C, Y represents C or T, and R represents A or G.
In one embodiment of the present invention, the gene chip is prepared as follows:
the designed specific probe sequences of each genotype are delivered to Shanghai Biometrics Limited company for synthesis, probes do not need to be additionally modified and swing arms are added, each probe is diluted to 100 ng/mu L by double distilled water to serve as storage concentration, each storage concentration probe is diluted to 10 ng/mu L by the double distilled water during spotting to serve as working concentration, a commercial gene chip spotting instrument is used for spotting on a square nylon membrane (a chip array is shown in figure 1), then the chip is placed into an ultraviolet instrument, the probe faces upwards, ultraviolet crosslinking reaction is carried out for 10-15min, and then the chip is shielded from light for 2h, so that the probes and a carrier are fully crosslinked to be firmly connected, the chip is stored at 4 ℃ for later use, and the optimal dilution concentration of the probes is 6 ng/mu L finally obtained through verification of different probe concentration gradients.
In a fifth aspect, the invention provides a kit containing the primers shown in SEQ ID NO. 1-4, the specific probes shown in SEQ ID NO. 5-20, the combination of the primers and the probes, or the gene chip.
Preferably, the kit further comprises a positive plasmid template, wherein the positive plasmid template is a vector carrying ompA gene fragments corresponding to A, B, C, D, E, E/B, F, WC and M56 genotypes respectively.
More preferably, the cloning vector is pMD 18-T.
In one embodiment of the present invention, to ensure the accuracy of the detection of the chip of the present invention, the kit comprises a set of plasmid standard reference substances used in combination with the chip. The plasmid standard reference substance is composed of recombinant plasmids of ompA genes of a standard strain VS1 of the Chlamydia psittaci genotype A, a standard strain CP3 of the B genotype, a standard strain GR9 of the C genotype, a standard strain NJ1 of the D genotype, a standard strain CPMN of the E genotype, a standard strain WS/TR/E30 of the E/B genotype, a standard strain VS225 of the F genotype, a standard strain WC of the WC genotype and a standard strain M56 of the M56 genotype. The recombinant plasmid was prepared from the pMD18-T plasmid. Specifically, the plasmid standard reference substances comprise the following 9 plasmid standard substances:
the genotype of Chlamydia psittaci A is pGM-T-VS1ompA plasmid standard;
the Chlamydia psittaci genotype B is pGM-T-CP3ompA plasmid standard;
the C genotype of the Chlamydia psittaci is pGM-T-GR9ompA plasmid standard;
the genotype D of the Chlamydia psittaci is pGM-T-NJ1ompA plasmid standard;
the Chlamydia psittaci E genotype is pGM-T-CPMN ompA plasmid standard;
the Chlamydia psittaci EB genotype is pGM-T-WS/TR/E30ompA plasmid standard;
the F genotype of the Chlamydia psittaci is pGM-T-VS225ompA plasmid standard;
the Chlamydia psittaci WC genotype is pGM-T-WC ompA plasmid standard;
chlamydia psittaci M56 genotype pGM-T-M56ompA plasmid standard.
The chip provided by the invention can detect and judge whether the strain to be detected is the psittaci chlamydia, if the strain is the psittaci heat positive strain, the genotype can be directly identified, and the genotype specificity detection probe of the chip comprises all 9 genotypes under C.psittaci species: chlamydia psittaci genotype A, genotype B, genotype C, genotype D, genotype E/B, genotype F, genotype WC and genotype M56.
In a sixth aspect, the invention provides a primer shown in SEQ ID NO. 1-4, a specific probe shown in SEQ ID NO. 5-20, a combination of the primer and the probe, and any one of the following applications of the gene chip or the kit:
1) chlamydia psittaci genotype detection for non-diagnostic purposes;
2) the kit is used for identifying the psittacosis serovar type.
Further, the application comprises the steps of:
1) extracting DNA in a sample;
2) carrying out PCR amplification reaction by using the primer by using the DNA extracted in the step 1) as a template;
3) and hybridizing the PCR amplification product with the specific probe, introducing a streptavidin-horseradish peroxidase system into the hybridization reaction, and adding a TMB substrate in the color development process to realize visual detection.
Preferably, the 5' end of the primer of the present invention is modified by Biotin.
Specifically, step 1) uses a commercial bacterial genome extraction kit to extract a nucleic acid sample of pathogenic bacteria to be detected.
And step 2) utilizing a double PCR reaction system of a first pair of primers (C.psitt _ omp _ f1 and C.psitt _ omp _ r1) and a second pair of primers (C.psitt _ omp _ f2 and C.psitt _ omp _ r2) to simultaneously amplify and obtain a target sequence of the chip, thereby improving the detection efficiency of the chip. The target sequences of the chip probe, namely VD1-VD2 and VD3-VD4 of ompA gene, are amplified. Preferably, the final concentration of the primer is 0.5. mu.M. The PCR product was detected by agarose gel electrophoresis, and the bands of interest were 418bp and 570bp, respectively.
Preferably, the PCR amplification conditions are: pre-denaturation at 96 ℃ for 1 min; 30s at 94 ℃; 30s at 55 ℃; 72 ℃ for 30 s; a total of 40 cycles; extending for 4min at 72 ℃; storing at 4 ℃.
Step 3), chip hybridization and color development:
the gene chip kit comprises various working solutions which are matched and used in the chip reaction process, and comprises: hybridization solution A, washing solution B, washing solution C, binding solution D, color development solution (W4 solution).
Prepare chip working solution with M1, M2 and M3 solutions (table 1): hybridization solution A, washing solution B and washing solution C (GeneChip kit reagents M1, M2, M3, W4, available from Biotech Co., Ltd., Shanghai province). The W4 liquid in the gene chip matching reagent is a precipitation type TMB color development liquid used in the chip color development reaction process.
TABLE 1
(1) Performing denaturation and hybridization, namely uniformly mixing PCR products and hybridization solution (A solution) in a volume ratio of 50 mu L: 1m L, placing the mixture into a metal bath at 96 ℃, performing denaturation for 4min, placing the mixture into an ice box, cooling for 2min, placing the gene chip into a hybridization box, adding the hybridization solution onto the surface of a chip array, and performing hybridization reaction for 1h at 60 ℃;
(2) color development and result interpretation of gene chip
1) Washing 1, after hybridization, discarding the hybridization solution in the hybridization box, rinsing with washing solution (B solution) preheated at 47 ℃ for one time, washing with washing solution (B solution) preheated at 47 ℃ for 10min, and repeating twice;
2) preparing binding solution (streptavidin-horse radish peroxidase (SA-HRP)) 2m L binding solution D in a volume ratio of solution A to 1 mu L to 2000 mu L, adding the binding solution D into a gene chip hybridization box, and incubating the hybridization box with the chip for 15min at room temperature;
3) washing 2, namely removing the binding solution D, taking out the gene chip, leaching the chip twice with the hybridization solution A at room temperature, and leaching the chip twice with a washing solution C at room temperature;
4) developing color by adding 400 mu L W4 solution (precipitation type TMB developing solution) on the surface of the chip, incubating for 2-4min at room temperature, then removing the developing solution, forming blue precipitation particles by the TMB under the catalysis of HRP, namely, developing quality control sites, positive control sites and captured target gene sites present blue spots, and the background of the chip presents white.
According to the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention combines the principle and the advantage of a gene chip and the advantage of a TMB precipitation type visual color development system according to the diversity and the regularity of each genotype of the C.psittaci on the ompA gene, obtains a high conservative fragment and a high variation fragment among genotypes by analyzing and screening the C.psittaci ompA gene sequence, obtains a primer and a genotype specific probe, establishes a visual color development oligonucleotide probe gene chip, and realizes the purpose of detecting and directly judging the genotype of the psittaci chlamydia.
Drawings
FIG. 1 is a schematic diagram of the spotting matrix of the Chlamydia psittaci genotype-specific gene chip in example 1 of the present invention.
FIG. 2 is an electrophoresis banding chart of the products obtained after the double PCR amplification of the gene sample to be tested in example 1 of the present invention with specific primers (C.psitt _ omp _ f1 and C.psitt _ omp _ r 1; C.psitt _ omp _ f2 and C.psitt _ omp _ r 2). Wherein: m is 2000bp marker; chlamydia psittaci 6BC strain (genotype A); chlamydia psittaci genotype F plasmid; chlamydia trachomatis; 4, chlamydia pneumoniae; 5, abortion chlamydia; chlamydia psittaci strain CP3 (genotype B); 7, escherichia coli; 8, salmonella.
FIG. 3 is the experimental map of verifying the specificity of the gene chip for each plasmid standard of 9 Chlamydia psittaci genotypes in example 2 of the present invention.
FIG. 4 is a diagram showing the detection of Chlamydia psittaci genotype-specific detection gene chip assay by the cell culture strain (A) and the clinically positive disease substance (B) in example 3 of the present invention.
FIG. 5 is a diagram showing the results of the specific detection of the gene chip in example 4 of the present invention.
Detailed Description
Unless otherwise indicated, the examples follow conventional experimental conditions, such as, for example, the Molecular Cloning handbook of Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a L laboratory Manual,2001), or conditions as recommended by the manufacturer's instructions.
Example 1 screening of Chlamydia psittaci genotype-specific probes and primers
1. Material
Chlamydia probes and primers: the synthetic method is carried out by the company of Token's bioengineering (Shanghai).
A gene chip sample application instrument,
PersonalArrayerTM16 personal spotter: provided by biotechnology limited of boao crystal classic, beijing.
Extraction kit of bacterial nucleic acid: total gold (Beijing) Biotechnology Ltd extraction Kit (EasyPureBacteria Genomic DNA Kit, cat # EE 161-01).
Nylon Membranes (Nylon Membranes, posively charged, Roche corporation): purchased from Sigma-Aldrich.
Horseradish peroxidase-labeled streptavidin (streptavidin-HRP): purchased from Thermo FisherScientific, inc.
Gene chip kit (M1, M2, M3, W4) was purchased from Biotechnology Ltd of Shanghai province. Preparing a chip working solution according to a proportioning specification (table 1): hybridization solution A, washing solution B and washing solution C. The W4 liquid in the gene chip matching reagent is a precipitation type TMB color developing liquid used in the color developing process of the chip.
2. Method of producing a composite material
The screening process of the chlamydia psittaci genotype specific primers and probes is as follows:
the ompA genome of all strains of C.psittaci was retrieved and downloaded in GenBank (https:// www.ncbi.nlm.nih.gov/GenBank /) on the NCBI website, including the genomes of 69 strains (Table 2).
TABLE 2 Chlamydia psittaci Strain names
The ompA genes of the genomes of all 69 strains of c.psittaci above were aligned and screened using the MegAlign software: according to the characteristics of the ompA gene, the ompA gene is subjected to segmental analysis, and VD1, VD2, VD3 and VD4 regions of all ompA genes of the Ipomoea batatas are respectively compared with five conservative regions among the VD regions.
Wherein, the screening of VD1, VD2, VD3 and VD4 regions aims at searching the "most hypervariable region". After a series of comparisons, a batch of Chlamydia psittaci genotype specific primary screening probes are preliminarily screened out, and the total number is 36. All the primary screening probes are spotted to form a gene chip, and the gene chip is detected by actual standard genome samples so as to analyze, verify and evaluate the efficiency of each primary screening probe. And (3) detecting the effectiveness of the primary screening probes by using each standard genome and standard plasmid of all genotype strains of the Chlamydia psittaci, and eliminating non-specific hybridization, low-efficiency hybridization and low-sensitivity probes, so as to finally obtain the most effective C.psittaci genotype specific probe which is practically available. Through a large number of practical standard genome sample verification analyses, a hypervariable region located in a VD1-VD2 region and a hypervariable region located in a VD3-VD4 region of an ompA gene are finally found, and the two C.psittaci hypervariable regions (VD1-VD2 region and VD3-VD4 region) are characterized in that the variation of nucleotide base sites presents typical regularity aiming at genotypes, namely, the difference between the genotypes is large, the conservation degree in the genotypes is good, and the corresponding probes have good reaction efficiency in practical detection verification and have high specificity and sensitivity. Therefore, the two sequences are used as the screening region of the genotype specific probe to finally obtain a set of optimized Chlamydia psittaci genotype specific probes, and the sequences of the probes are shown in SEQ ID NO:5-20 (Table 4).
Thus, the ompA gene conserved regions at the upper and lower reaches of the two sections of 'most high mutation region' sequences are respectively screened and compared to obtain two pairs of universal primers, wherein the first pair of primers: psitt _ omp _ f1 and c.psitt _ omp _ r1, the size of the target fragment of the primer is 418bp, and the sequence of the primer is: 1-2 of SEQ ID NO; a second pair of primers: psitt _ omp _ f2 and c.psitt _ omp _ r2, the size of the target fragment of the primer is 570 bp; the sequence of the primer is as follows: 3-4 (Table 3) SEQ ID NO.
Simultaneously designing a chromogenic quality control probe of the gene chip as well as a positioning probe, a positive control probe and a negative control probe: the sequence complementary to the primers C.psitt _ omp _ f1 and C.psitt _ omp _ f2 served as positive control probes, the hybridization solution served as negative control probes, the chromogenic control probe-cum-localization probe was 20T sequence, and Biotin (Biotin) was ligated to the 5' end. The color development quality control probe is also used as a positioning probe.
TABLE 3 Chlamydia psittaci-specific amplification primers
TABLE 4 Chlamydia psittaci genotype specific oligonucleotide probe sequences
The information on the degenerate bases involved in the above sequences is as follows: degenerate base N represents hypoxanthine nucleotide I; degenerate base W represents a nucleotide: a, T; degenerate base representation M represents nucleotides A, C; degenerate base Y represents nucleotide C, T; the degenerate base R represents the nucleotides A, G.
Example 2 preparation of Chlamydia psittaci genotype-specific Gene chip
In this embodiment, the optimal reaction system for manufacturing the chip is finally optimized by preparing the chip from the optimal primer and the probe finally obtained in embodiment 1 and optimizing the hybridization condition and the color development condition of the chip, so as to ensure that the detection result of the chip is optimal.
1. Material
Chlamydia probes and primers: the synthetic method is carried out by the company of Token's bioengineering (Shanghai).
A gene chip sample application instrument,
personal Arrayer 16 personal spotter: provided by biotechnology limited of boao crystal classic, beijing.
Extraction kit of bacterial nucleic acid: total gold (Beijing) Biotechnology Ltd extraction Kit (EasyPureBacteria Genomic DNA Kit, cat # EE 161-01).
Nylon Membranes (Nylon Membranes, posively charged, Roche corporation): purchased from Sigma-Aldrich.
Horseradish peroxidase-labeled streptavidin (streptavidin-HRP): purchased from Thermo FisherScientific, inc.
Gene chip kit (M1, M2, M3, W4) was purchased from Biotechnology Ltd of Shanghai province. Preparing a chip working solution according to a proportioning specification (table 1): hybridization solution A, washing solution B and washing solution C. The W4 liquid in the gene chip matching reagent is a precipitation type TMB color developing liquid used in the color developing process of the chip.
2. Method of producing a composite material
2.1 preparation Process of Chlamydia psittaci genotype specific Gene chip and Probe array
The finally determined probes and primers, committed Bio-engineering (Shanghai) Co., Ltd, were synthesized without adding a swing arm and special modification, Biotin modification was performed at 5' of each primer with reference to Table 3. the synthesized primers and dry probe powder were stored in a refrigerator at-20 ℃ for use, the centrifuge tube containing the probes (dry powder) was placed in a centrifuge, centrifuged at 12000rpm for 5min, double distilled water was added to dilute each probe to a concentration of 100 ng/. mu. L as the storage concentration, and double distilled water was used to dilute the probes to a concentration of 10 ng/. mu. L as the working concentration during spotting.
The final probe array was identified as 5 × 5 array, see FIG. 1. the letters in the array represent the following:
| A1 | psittaci A genotype probe | SEQ ID NO:5 | F2 | Psittaci F genotype probe | SEQ ID NO:16 |
| A2 | Psittaci A genotype probe | SEQ ID NO:6 | F3 | C.psittaci F genotype probe | SEQ ID NO:17 |
| B | Psittaci B genotype probe | SEQ ID NO:7 | WC1 | Psittaci WC genotype probe | SEQ ID NO:18 |
| C1 | Psittaci C genotype probe | SEQ ID NO:8 | WC2 | Psittaci WC genotype probe | SEQ ID NO:19 |
| C2 | Psittaci C genotype probe | SEQ ID NO:9 | M56 | Psittaci M56 genotype probe | SEQ ID NO:20 |
| D1 | Psittaci D genotype probe | SEQ ID NO:10 | PC1 | Positive control probe | SEQ ID NO:21 |
| D2 | Psittaci D genotype probe | SEQ ID NO:11 | PC2 | Positive control probe | SEQ ID NO:22 |
| E1 | Psittaci E genotype probe | SEQ ID NO:12 | SC | Color development quality control probe | SEQ ID NO:23 |
| E2 | Psittaci E genotype probe | SEQ ID NO:13 | NC | Negative probe | Hybrid liquid |
| E/B | Psittaci E/B genotype probe | SEQ ID NO:14 | |||
| F1 | Psittaci F genotype probe | SEQ ID NO:15 |
According to the probe array described in FIG. 1, a commercially available gene chip spotting instrument (Beijing Boo At Crystal Biotechnology Co., Ltd.) is used to spot samples on a square nylon membrane in a contact or spray manner according to a set program, or in the absence of a spotting instrument, a gene chip can be manually prepared by adjusting to 0.2 μ L with a 10 μ L pipette, absorbing the corresponding diluted probe, spotting on the square nylon membrane (the spotting array is shown in FIG. 1), placing the chip on an ultraviolet instrument after completing spotting, performing a crosslinking reaction for 10min, keeping out of the sun for 2h to fully crosslink the probe and the carrier, and then storing for later use.
2.2 Dual PCR reaction specific amplification
The extraction of nucleic acid of pathogenic Bacteria was carried out by using a bacterial genome rapid extraction Kit (easy pure bacterial Genomic DNA Kit, cat # EE161-01) of the entire gold (Beijing) Biotechnology Ltd, according to the instruction.
The double PCR reaction system (Table 5) of the first pair of primers (C.psitt _ omp _ f1 and C.psitt _ omp _ r1) and the second pair of primers (C.psitt _ omp _ f2 and C.psitt _ omp _ r2) is used for improving the detection efficiency of the chip and amplifying target sequence-two sections of target sequence regions VD1-VD2 and VD3-VD4 of ompA gene of the chip probe. The preferred final concentration of primers is 0.5. mu.M. The double PCR products were detected by agarose gel electrophoresis, and the bands of interest were 418bp and 570bp, respectively.
The synthesized primer dry powder is placed in a centrifuge and centrifuged for 5min at 12000rpm, double distilled water is added to dilute the primer dry powder to the concentration of 100 mu M as the storage concentration, and 10 mu L is taken to dilute the primer dry powder to 10 mu M as the working concentration during PCR.
TABLE 5 Dual PCR System
| Components | Volume of | Final concentration |
| Form panel | 2.0μL | |
| C.psitt_omp_f1 | 1μL | 0.5μM |
| C.psitt_omp_r1 | 1μL | 0.5μM |
| C.psitt_omp_f2 | 1μL | 0.5μM |
| C.psitt_omp_r2 | 1μL | 0.5μM |
| PCR SuperMix | 25μL | |
| ddH2O | 19μL | |
| Total volume | 50μL |
The PCR amplification reaction conditions are as follows: pre-denaturation at 96 ℃ for 1 min; 30s at 94 ℃; 30s at 55 ℃; 72 ℃ for 30 s; a total of 40 cycles; extending for 4min at 72 ℃; storing at 4 ℃.
After the reaction is finished, the PCR product is detected by 1.5% agarose gel electrophoresis, and the target band sizes are 417bp and 570 bp. FIG. 2 is the electrophoresis chart of the PCR amplified product of the selected Chlamydia strain gene sample. The PCR product was stored at 4 ℃ or subjected to the next experiment.
2.3 Gene chip hybridization and color development protocol
2.3.1 establishment of chip hybridization System
(1) Performing denaturation and hybridization, namely uniformly mixing PCR products, namely hybridization solution (A solution) 50 mu L: 1m L in volume ratio, placing the mixture into a metal bath at 96 ℃, performing denaturation for 4min, placing the mixture into an ice box, cooling for 2min, placing the gene chip into a hybridization box, adding the hybridization solution onto the surface of a chip array, and performing hybridization reaction for 1h at 60 ℃;
(2) color development and result interpretation of gene chip
1) Washing 1, after hybridization, discarding the hybridization solution in the hybridization box, rinsing with washing solution (B solution) preheated at 47 ℃ for one time, washing with washing solution (B solution) preheated at 47 ℃ for 10min, and repeating twice;
2) preparing binding solution of streptavidin-horse radish peroxidase (SA-HRP) and 2m L binding solution D in the volume ratio of solution A to 1 μ L to 2000 μ L, adding the binding solution into a gene chip hybridization box, and incubating the hybridization box with the chip at room temperature for 15 min;
3) washing 2, namely removing the binding solution D, taking out the gene chip, leaching the chip twice with the hybridization solution A at room temperature, and leaching the chip twice with a washing solution C at room temperature;
4) developing color by adding 400 mu L W4 solution (precipitation type TMB developing solution) on the surface of the chip, incubating for 2-4min at room temperature, then removing the developing solution, forming blue precipitation particles by the TMB under the catalysis of HRP, namely, developing quality control sites, positive control sites and captured target gene sites present blue spots, and the background of the chip presents white.
2.3.2 optimization of hybridization reaction System
The specificity and sensitivity of the gene chip of the invention can be ensured and improved by using the optimized hybridization reaction conditions, and the following conditions are optimized by the invention: the probe sample concentration, the proportion of the hybridization reaction solution, the hybridization reaction temperature and time, the washing conditions, the incubation temperature and time of the binding solution, the color reaction time and other parameters are explored, and the exploration and screening of the optimal conditions are shown in Table 6.
TABLE 6 optimization of conditions for hybridization reactions on Gene chips
The optimal reaction system of the chip finally and preferably selected is that the optimal sample application concentration of the probe is 6 ng/mu L, the optimal proportion ratio of the reaction liquid in the hybridization process is 50 mu L: 1m L, the optimal temperature of the hybridization reaction is 55 ℃, the optimal time is 60min, the washing condition is water bath 37 ℃, 3 times, the optimal time and temperature of incubation of the binding liquid are water bath 37 ℃ and 20min, the optimal color development condition is room temperature and dark for 2-4min, the chip reaction is carried out under a series of optimal system conditions, the detection result of the chip reaches the optimal, and the specificity, repeatability and stability of the chip are good.
EXAMPLE 3 construction of Chlamydia psittaci plasmid Standard reference
In the embodiment, a set of matched plasmid standard reference substances of the gene chip is established, so that the opportunity of purifying a standard genome from a chlamydia pathogen is reduced, the infection probability is reduced, and the efficiency of the gene chip is more conveniently verified and detected; meanwhile, the kit is also used as a standard reference substance matched with the chlamydia species specificity chip detection kit, and a positive control is established in the clinical sample detection process.
1. Materials:
standard strains for each genotype:
| genotype(s) | Standard strains |
| A | VS1 |
| B | CP3 |
| C | GR9 |
| D | NJ1 |
| E | CPMN |
| F | VS225 |
| E/B | WS/TR/E30 |
| M56 | M56 |
| WC | WC |
The laboratory has deposited Chlamydia psittaci A, B, C and a standard strain or genome of D genotype.
Bacterial genome DNA extraction kit: easypore bacteriogenic DNA Kit of beijing holotype gold biotechnology limited, cat no: EE 161-01.
Plasmid DNA visual extraction kit: plasmid MiniPrep Kit, cat No.: EM 101-01.
pEASY-T1Cloning Kit: beijing Quanjin Biotechnology Ltd, cat #: CT101-01, and the cloning vector contained in the rapid cloning kit is pMD18-T vector.
Micro-spectrophotometer: nano-300, Hangzhou Osheng instruments, Inc. 2. The method comprises the following steps:
2.1 genome extraction: respectively culturing and purifying the owned strains, extracting genome, performing PCR target sequence amplification by using a specific primer aiming at the ompA gene of the Chlamydia psittaci, and performing agarose gel electrophoresis detection on a PCR result. The agarose gel recovery kit recovers and purifies the PCR product and submits to sequencing comparison with Biotechnology engineering (Shanghai) GmbH to achieve double validation.
The other E, F, E/B, M56 and WC genotypes do not obtain standard strains or genomes, and in order to meet the experimental requirements, the invention synthesizes the ompA sequence samples of the five standard strains of the C.psittaci genotypes according to the ompA gene deoxynucleotide sequences of the CPMN strain (E genotype) of the Chlamydia psittaci, the VS225 strain (F genotype) of the Chlamydia psittaci, the WS/TR/E30 strain (E/B genotype) of the Chlamydia psittaci, the M56 strain (M56 genotype) of the Chlamydia psittaci and the WC strain (WC genotype) which are disclosed on NCBI website GenBank (https:// www.ncbi.nlm.nih.gov/GenBank /), and stores the samples after dilution according to certain concentration.
2.2 construction and preparation of cloning plasmid: the Cloning vector was constructed using the pEASY-T1Cloning Kit.
The cloning reaction system is shown in table 7:
TABLE 7 cloning reaction System
| Composition (I) | Volume of |
| PCR Product | 0.5-4μL |
| pEASY-T1 Cloning Vector | 1μL |
The reagents are added into a centrifuge tube and then mixed gently, and then the mixture is reacted for 5 minutes at room temperature (20 ℃ -37 ℃). After the reaction was completed, the mixture was put on ice.
Adding the ligation product into 50 mu L Trans1-T1 competent cells, flicking and mixing uniformly, ice-bathing for 20-30 min, heat-shocking in 42 ℃ water bath for 30 sec, immediately placing on ice for 2min, taking 250 mu L-L B culture medium, culturing at 200rpm and 37 ℃ for 1h, taking 200 mu L bacterial liquid, uniformly spreading on a plate containing IPTG and X-gal, and carrying out screening culture.
2.3 detection of Positive cloned plasmid and extraction of plasmid DNA
Extracting plasmid DNA from the sequencing target fragment-containing monoclonal bacterial liquid by using a plasmid miniprep kit, measuring the concentration of the extracted plasmid by using a microspectrophotometer, calculating the copy number according to a formula, diluting the obtained plasmid into a gradient of 1 × 10 without DNA enzyme water6copies/μL、1×105copies/μL、1×104copies/μL、1×103copies/μL、1×102copies/μL、1×101copies/. mu. L, subpackaged and stored at-20 ℃ for later use.
4. Finally, the plasmid standard reference substances for obtaining the 9 genotype standard strains of Chlamydia psittaci are successfully constructed as follows:
the genotype of Chlamydia psittaci A is pGM-T-VS1ompA plasmid standard;
the Chlamydia psittaci genotype B is pGM-T-CP3ompA plasmid standard;
the C genotype of the Chlamydia psittaci is pGM-T-GR9ompA plasmid standard;
the genotype D of the Chlamydia psittaci is pGM-T-NJ1ompA plasmid standard;
the Chlamydia psittaci E genotype is pGM-T-CPMN ompA plasmid standard;
the Chlamydia psittaci EB genotype is pGM-T-WS/TR/E30ompA plasmid standard;
the F genotype of the Chlamydia psittaci is pGM-T-VS225ompA plasmid standard;
the Chlamydia psittaci WC genotype is pGM-T-WC ompA plasmid standard;
chlamydia psittaci M56 genotype pGM-T-M56ompA plasmid standard.
Through multiple verification, the constructed plasmid standard is accurate, and FIG. 3 is an experimental result diagram of the gene chip for verifying the plasmid standard of 12 chlamydia species.
The plasmid standard substance constructed by the invention has good effect in chip detection, and the specificity among various plasmid standard substances reaches the standard, and can be used as a standard basis for chip verification and sample detection and as a reference substance for specificity and sensitivity detection.
Example 4 sample detection and conformity evaluation of chip for Chlamydia psittaci species-specific detection
This example was used to detect Chlamydia psittaci in cell culture strain specimens, clinically collected throat swabs, cloacal swabs, alveolar lavage fluid, placental membranes, vaginal secretions and to identify the genotype of Chlamydia psittaci using the chip provided by the present invention.
1. Materials:
bacterial genome DNA extraction kit: all-purpose gold biotechnology (Beijing) Inc. EasyPure bacteriogenic DNA Kit, cat #: EE 161-01.
Gene chip: the kit prepared in example 1.
Reference group: chlamydia ompA gene sequencing (Biotechnology engineering (Shanghai) Co., Ltd.).
Sample source:
(1) standard genomic sources: 15 cell culture strain culture dishes (containing Chlamydia psittaci: 6BC strain, CP3 strain, NJ1 strain, GR9 strain, Chlamydia trachomatis: D/UW-3/CX strain, Chlamydia pneumoniae: N16 strain, Chlamydia abortus: B577 strain, Chlamydia murinus: Mopn strain, and negative pure cell culture).
(2) Clinical samples: throat swabs, cloaca swabs, alveolar lavage fluid, fetal membranes, vaginal secretion samples.
2. The method comprises the following steps:
and (3) genome purification: cell culture strain specimen, throat swab, cloaca swab, alveolar lavage fluid, fetal membrane and vaginal secretion sample, which are diluted by proper sterilized normal saline, can be directly used for extracting nucleic acid sample by using the kit; after the nucleic acid preparation is completed, the detection of a gene sample to be tested and the identification of the chlamydia species attribute can be performed using the chlamydia species-specific gene chip of the present invention as described in example 1.
Randomly selecting 15 parts of cell culture strain culture dishes (containing 6BC strain, CP3 strain, NJ1 strain and GR9 strain of Chlamydia psittaci, D/UW-3/CX strain, N16 strain, B577 strain, mouse Chlamydia, Mopn strain and negative pure cell culture) cultured by irrelevant personnel of a gene chip for blind test; 80 clinical collected pathological materials (including throat swabs, alveolar lavage fluid, fetal membranes and vaginal secretions) are selected for detection, and the detection is carried out while results of a sequencing method are compared, so that the accuracy and the conformity of the gene chip are verified.
3. As a result:
the Chlamydia psittaci genotype specific gene chip of the invention is used for detecting a standard strain cell culture sample and a clinical suspected disease sample, and is compared with a sequencing result of corresponding Chlamydia ompA gene of biological engineering (Shanghai) corporation, and the detection results of the chip method and the sequencing method are shown in Table 8.
TABLE 8 sample detection and compliance verification of Chlamydia species-specific Gene chips
The coincidence rate of the statistical chip method detection samples is as follows:
| sample type | Sample size | Coincidence rate of gene chip detection result and sequencing result |
| Cell culture strains | 15 |
100％ |
| Clinical collection of pathological samples | 45 portions of | 97.78％ |
FIG. 4 shows the results of gene chip detection of cell culture strains and genomes extracted from clinical suspected disease.
4. The statistics of the coincidence rate of the detection result and the sequencing result of the gene chip are as follows: the chip aims at a clean cell culture sample with a simpler background, the detection accuracy and the conformity can reach 100 percent, and the chip detection accuracy and the conformity can reach 97.78 percent in the aspect of clinical collection of pathological material detection with a complex background and relatively low pathogen load. Therefore, the accuracy and the conformity of the Chlamydia psittaci genotype specific gene chip disclosed by the invention are relatively high, and the chip can be used for laboratory diagnosis and research and can also be used for accurately detecting samples in practice.
Example 5 evaluation of specificity of Chlamydia psittaci species-specific detection chip
This example was used for evaluating the specificity of a chlamydia species-specific detection chip. Selecting Chlamydia abortus, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia suis, Chlamydia livestock, Escherichia coli, Salmonella, Mycoplasma pneumoniae and H9N2Avian influenza virus was used as a specificity validation sample for c. The prepared Chlamydia psittaci genotype specific diagnosis gene chip is used for detection, and the specificity of the gene chip is verified. After extracting virus RNA from a virus sample, firstly, the RNA is reversely transcribed into cDNA, and then PCR and chip detection are carried out.
1. Materials:
the strain source is as follows:
chlamydia psittaci strain CB3 (genotype A), Chlamydia psittaci strain CP3 (genotype B), Chlamydia psittaci strain 9N (genotype D), Chlamydia pneumoniae, Chlamydia trachomatis, Mycoplasma pneumoniae, Escherichia coli, H9N 2.
Bacterial genome DNA extraction kit: easy purebacteria Genomic DNA Kit of total gold biotechnology (beijing) ltd, cat no: EE 161-01.
Gene chip: the kit prepared in example 2.
2. The method comprises the following steps:
detecting by using the chlamydia species specificity gene chip under the optimized condition, and verifying the specificity of the gene chip.
3. As shown in FIG. 5, the above-mentioned common pathogens were all detected as negative, indicating that the gene chip had good specificity.
The gene chip has good specificity, can detect the genotype specificity of the chlamydia psittaci under the condition of aiming at each single strain or simulating clinical actual mixed infection of various chlamydias, can still distinguish and identify the genotype of the chlamydia psittaci, achieves good detection effect, and is suitable for clinical actual sample analysis.
Example 6 sensitivity analysis of Chlamydia psittaci species-specific detection chip
This example was used to evaluate the sensitivity of a Chlamydia psittaci gene-specific gene chip.
1. Materials:
plasmid standard: the plasmid prepared in example 3;
gene chip: the chip prepared in example 2;
2. method of producing a composite material
The plasmid standards of each genotype of Chlamydia psittaci prepared in the above-mentioned example 3 were each set with a dilution concentration gradient to examine the sensitivity and minimum detection limit of the gene chip. Each concentration is 106copies/μL、105copies/μL、104copies/μL、103copies/μL、102copies/μL、101And (4) performing chip detection by using the optimized chip reaction system, namely copies/mu L.
3. Results
The result shows that the detection limit of the Chlamydia psittaci genotype detection chip of the invention on each genotype of Chlamydia psittaci A, B, C, D, E, E/B, F, WC and M56 is 2 × 103copies/reaction (2 refers to the amount of plasmid template added during PCR reaction), so the lowest detection limit of the gene chip of the invention for detecting various chlamydiae is 2 × 103copies/reaction, the performance is better.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (12)
1. The primer for detecting the genotype of Chlamydia psittaci is characterized by comprising 2 pairs of primers, a primer pair 1 and a primer pair 2, wherein the sequences are respectively shown as SEQ ID NO. 1-2 and SEQ ID NO. 3-4.
2. A probe set for the genotype detection of chlamydia psittaci, said probe set comprising:
chlamydia psittaci A genotype specific probe with sequence shown in SEQ ID NO 5-6;
a Chlamydia psittaci genotype B specific probe, the sequence of which is shown in SEQ ID NO. 7;
the Chlamydia psittaci C genotype specific probe has the sequence shown in SEQ ID NO 8-9;
chlamydia psittaci D genotype specific probe with sequence shown in SEQ ID NO 10-11;
chlamydia psittaci E genotype specific probe with sequence shown in SEQ ID NO 12-13;
chlamydia psittaci E/B genotype specific probe, the sequence is shown in SEQ ID NO. 14;
chlamydia psittaci F genotype specific probe with sequence shown in SEQ ID NO 15-17;
the Chlamydia psittaci WC genotype specific probe has the sequence shown in SEQ ID NO 18-19;
chlamydia psittaci M56 genotype specific probe, the sequence is shown in SEQ ID NO: 20.
3. A primer and probe combination for Chlamydia psittaci genotype detection, comprising the primer of claim 1 and the probe combination of claim 2.
4. A chip for the genotype test of Chlamydia psittaci, comprising a chip carrier and the specific probe as claimed in claim 2.
5. The chip of claim 4, further comprising 2 positive control probes, the sequences of which are shown in SEQ ID NO 21-22; 1 quality control probe with the sequence shown in SEQ ID NO. 23; and a control probe which is a spotting diluent.
6. The chip of claim 4 or 5, wherein the spotting concentration of the probes on the chip is 6ng/μ L.
7. A kit comprising the primer according to claim 1, the probe combination according to claim 2, the primer and probe combination according to claim 3, or the chip according to any one of claims 4 to 6.
8. The kit of claim 7, further comprising a positive plasmid template, wherein the positive plasmid template is a vector carrying ompA gene fragments corresponding to A, B, C, D, E, E/B, F, WC, and M56 genotypes, respectively.
9. The kit of claim 8, wherein the cloning vector used for the positive plasmid template is pMD 18-T.
10. Use of the primers according to claim 1, the probe combination according to claim 2, the primer and probe combination according to claim 3, the chip according to any one of claims 4 to 6, or the kit according to any one of claims 7 to 9 for the detection of chlamydia psittaci genotypes for non-diagnostic purposes.
11. Use according to claim 10, characterized in that it comprises the following steps:
1) extracting DNA in a sample;
2) carrying out PCR amplification reaction by using the primer by using the DNA extracted in the step 1) as a template;
3) and hybridizing the PCR amplification product with the specific probe, introducing a streptavidin-horseradish peroxidase system into the hybridization reaction, and adding a TMB substrate in the color development process to realize visual detection.
12. The use of claim 11, wherein the 5' end of the primer is modified by Biotin;
step 2) PCR amplification reaction conditions are as follows: 1 minute at 96 ℃; 30 seconds at 94 ℃, 30 seconds at 55 ℃ and 30 seconds at 72 ℃ for 40 cycles; 72 ℃ for 4 minutes.
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| CN101579521A (en) * | 2009-05-13 | 2009-11-18 | 中国农业科学院兰州兽医研究所 | Abortus Chlamydia psittaci vaccine and preparation method thereof |
| CN101608239A (en) * | 2009-03-26 | 2009-12-23 | 重庆大学 | Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof |
| CN103981252A (en) * | 2013-11-27 | 2014-08-13 | 宁夏农林科学院 | Method for rapid detection of bovine Chlamydia psittaci |
| WO2018175875A1 (en) * | 2017-03-23 | 2018-09-27 | Ohio State Innovation Foundation | Recombinant chlamydia-activated b cell platforms and methods of use thereof |
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| CN101608239A (en) * | 2009-03-26 | 2009-12-23 | 重庆大学 | Animal chlamydia multiple sleeve type PCR detection kit and detection method thereof |
| CN101579521A (en) * | 2009-05-13 | 2009-11-18 | 中国农业科学院兰州兽医研究所 | Abortus Chlamydia psittaci vaccine and preparation method thereof |
| CN103981252A (en) * | 2013-11-27 | 2014-08-13 | 宁夏农林科学院 | Method for rapid detection of bovine Chlamydia psittaci |
| WO2018175875A1 (en) * | 2017-03-23 | 2018-09-27 | Ohio State Innovation Foundation | Recombinant chlamydia-activated b cell platforms and methods of use thereof |
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