CN116732212B - Primer combination and application thereof in preparation of Galsen-Gingo nocardia complex group detection kit - Google Patents

Primer combination and application thereof in preparation of Galsen-Gingo nocardia complex group detection kit Download PDF

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CN116732212B
CN116732212B CN202311004261.6A CN202311004261A CN116732212B CN 116732212 B CN116732212 B CN 116732212B CN 202311004261 A CN202311004261 A CN 202311004261A CN 116732212 B CN116732212 B CN 116732212B
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李振军
徐帅
李臻鹏
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National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
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Abstract

The invention provides a primer combination and application thereof in preparing a Galsonyl-Xinghai nocardia complex group detection kit, wherein the primer combination comprises an upstream primer and a downstream primer for a polymerase chain amplification reaction, which are designed by taking NCTC10797_05230 of Galsonyl-Xinghai nocardia as a target gene, IU407_RS05750 of Ningxia nocardia as a target gene, and/or taking NOCYR_RS03685 of Yinchian nocardia as a target gene. The primer combination is used for differential diagnosis of George-Mecanum, ningxia Nocardia and Yinchuannocardia.

Description

Primer combination and application thereof in preparation of Galsen-Gingo nocardia complex group detection kit
Technical Field
The invention discloses a primer combination, and belongs to the technical field of nucleic acid.
Background
Nocardia genusNocardia) The bacterial strain is widely used in natural environment, is opportunistic pathogenic bacteria which are ignored for a long time, and is mainly used for people with low immunity and immunodeficiency. The bacteria mainly cause skin abscesses and lung abscesses, and sometimes also cause brain abscesses and even systemic disseminated diseases. Among nocardia capable of causing human infection, nocardia gambir @ of ganciclovirN. cyriacigeorgica) Severe pneumonia is more likely to occur, which can survive, proliferate in alveolar macrophages, and in turn induce lung injury. The strain with strong toxicity can also penetrate capillary endothelial cells and spread to brain parenchyma, secondary nervous system diseases, and cause headache, fever, epilepsy, hemiplegia, etc. Meanwhile, the Gelson groupSinuoka bacteria is also one of the most susceptible species to systemic spread infections, which can lead to serious consequences in patients with immunodeficiency or chronic pulmonary disease.
2022, based on the whole genome sequence discovery, the ganciclovir has high heterogeneity, is not a single strain, but consists of five species, collectively referred to as ganciclovir complex groupN. cyriacigeorgica complex), wherein, the Gelson-Xinghuoka bacterium and Ningxia-nocardia @N. ningxiensis) And YinchuannocardiaN. yinchuanensis) Is clinically common nocardia. The three species have different pathogenic characteristics and drug sensitivity modes, are easily confused with tuberculosis and other bacterial infectious diseases of the lung, have high diagnosis and treatment difficulty, are easy to delay treatment, and cause high death rate of hospitalization. The conventional 16S rRNA gene sequencing (the sequence similarity is more than 99.8%), mass spectrometry and other methods cannot effectively distinguish the sequences. Clinically, the technical bottleneck problem that the Gerssonchifolia nocardia, ningxia nocardia and Yinchuannocardia cannot be identified exists.
Therefore, the invention aims to carry out comparative genomics analysis on the George's Nocardia, ningxia's Nocardia and Yinchuan Nocardia by utilizing a bioinformatics analysis technology, screen a seed-specific gene as a target gene at a genome level, design a specific primer combination, and use the primer combination in a chimeric fluorescent dye qPCR technology to realize differential diagnosis on the George's Nocardia, ningxia's Nocardia and Yinchuan Nocardia.
Disclosure of Invention
In view of the above objects, the present invention provides, first, a primer combination for a polymerase chain amplification reaction, the combination comprising:
(1) Polymerase chain amplification reaction upstream primer and downstream primer designed by taking NCTC10797_05230 of Gelson's Nocardia as target gene;
(2) An upstream primer and a downstream primer of a polymerase chain amplification reaction designed by taking IU407_RS05750 of Ningxia nocardia as a target gene; and/or
(3) And (3) taking NOCYR_RS03685 of Nocardia guarantor as a target gene to design an upstream primer and a downstream primer of a polymerase chain amplification reaction.
The invention predicts genomic CDS based on Prodigal v2.3.6 and calculates non-redundant homologous gene datasets using CD-HIT v 4.6.6; merging all CDSs; performing BLAST on the non-redundant homologous data set and the combined CDS, wherein the threshold value is E-value 10 -5 Coverage is 60% and a flood genome dataset is obtained. If the gene is present in all target species but not in non-target species, it is a potential species-specific gene. The potential species-specific genes are extracted, NCBI nucleic acid sequence databases are aligned by using a Blastn program, and medium-specific genes of George's Saccharomycetes, ningxia's Nordic acid bacteria and Yinchun's Nordic acid bacteria are obtained by screening, and a person skilled in the art can design the upstream primer and the downstream primer based on the polymerase chain amplification reaction (PCR) of the 3 target genes according to the basic principles of molecular biology and conventional techniques for the target genes.
In a preferred embodiment, the sequences of the upstream primer and the downstream primer of the polymerase chain amplification reaction designed by taking NCTC10797_05230 of George's Nocardia as a target gene are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.
In another preferred embodiment, the sequences of the upstream primer and the downstream primer of the polymerase chain amplification reaction designed by taking IU407_RS05750 of Ningxia nocardia as a target gene are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
In a further preferred embodiment, the sequences of the upstream primer and the downstream primer of the polymerase chain amplification reaction designed by taking NOCYR_RS03685 of the Nocardia guarantor as a target gene are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.
In a preferred embodiment, the polymerase chain amplification reaction is a real-time fluorescent quantitative polymerase chain amplification reaction.
Secondly, the invention provides a kit containing the primer combination, and the kit is a chimeric fluorescent dye method real-time fluorescence quantitative polymerase chain amplification reaction.
In a preferred embodiment, the kit further comprisesTaqPolymerizationEnzymes, dntps, and fluorescent dyes.
In a preferred embodiment, the fluorescent dye is TB Green.
Finally, the invention also provides application of the primer combination in preparation of a detection kit for the composite group of the Gelson-Xinghai Nordic bacteria.
In a preferred embodiment, the complex population of ganciclovir comprises ganciclovir, ganciclovir and ganciclovir.
The PCR primer combination for specificity in the Gelson-Xinghua Nocardia, ningxia Nocardia and Yinchuannocardia provided by the invention has the amplification efficiency of 90.25% -96.15% and the detection lower limit of 10 in the specific application of qPCR 2 ~10 1 The copies/. Mu.L order shows excellent sensitivity. The detection results of the three nocardia bacteria are consistent with the detection results of the traditional separation culture method and the whole genome identification sequencing result, so that the invention can effectively identify the three nocardia bacteria. qPCR method specificity was assessed on nucleic acids from seven nocardia and seven clinically common respiratory pathogens. The results show that seven nocardia, 17 near non-target strains and seven clinically common respiratory pathogens do not produce specific amplification, and the specificity is 100%.
Drawings
FIG. 1 amplification curve of the qPCR of Nocardia galsonii;
FIG. 2 shows that the qPCR amplification product of Nocardia galsonii has a specific melting curve;
FIG. 3 standard curve of qPCR amplification of Nocardia galsonii;
FIG. 4 amplification curve of Ningeca bacteria qPCR;
FIG. 5 shows that the Ningeca bacteria qPCR amplification product has a specific melting curve;
FIG. 6 standard curve of Ningeca bacteria qPCR amplification;
FIG. 7 amplification curve of Sinonoka qPCR;
FIG. 8. The product of the Sinonoka qPCR amplification has a specific melting curve;
FIG. 9 standard curve of the qPCR amplification of Minnocardia guarantor.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.
The main reagent used in the invention is as follows:
genomic DNA purification kit (Wizard Genomic DNA Purification Kit) was purchased from Promega company, USA, qPCR reaction system mixture (TB Green)premix Ex Taq TM GC) was purchased from TAKARA corporation, japan. The remaining reagents were all conventional commercial reagents.
The main instrument used in the invention is as follows:
NanoDrop ND-1000 Spectrophotometer is manufactured by Thermo Fisher, inc. of America. The real-time fluorescence quantitative PCR instrument is manufactured by Roche company of Switzerland.
Standard strains and clinical strains used in the present invention:
the model strains and standard strains of nocardia guerbeta, nocardia melitensis, nocardia otophylli, nocardia novariscina, nocardia asiatica, nocardia brasiliensis and nocardia abscesses were purchased from the german collection of microbial strains. Ningxia nocardia and Yinchuan nocardia model strains are separated, identified and named in the laboratory and are preserved in Japanese microbiological bacterial collection center. The Nocardia phlegm model strain is presented by Beijing Korea and is preserved in Japanese microbiological culture collection center. Klebsiella pneumoniae, pseudomonas aeruginosa, streptococcus pneumoniae, haemophilus influenzae, and staphylococcus aureus were purchased from the american type culture collection. Nocardia clinical isolates were collected, identified and deposited for this laboratory. Other strains are derived from the China center for disease prevention and control, and specific information is shown in Table 1.
TABLE 1 Standard and clinical strains for detection
T : mode strain.
Genomic sequences used in the present invention:
the invention incorporates 94 genomic data including 63 ganciclovir, 11 ninja and 20 ganciclovir, and specific information is shown in table 2.
TABLE 2 genomic information incorporated by the invention
Specific gene screening:
genome CDS was predicted using Prodigal v2.3.6 and non-redundant homologous gene datasets were calculated using CD-HIT v 4.6.6. All CDSs are combined. Performing BLAST on the non-redundant homologous data set and the combined CDS, wherein the threshold value is E-value 10 -5 Coverage is 60% and a flood genome dataset is obtained. If the gene is present in all target species but not in non-target species, it is a potential species-specific gene. Potential species-specific genes were extracted and aligned to the NCBI nucleic acid sequence database using the Blastn program. If the gene is not present in other species (E-value is greater than 10 -5 Coverage less than 20%), the species-specific genes were identified (see table 3). After Blast comparison, the number of the specific genes is four of the Gerson Nocardia, nine of the Ningxia Nocardia and ten of the Yinchuannocardia respectively, and most of the genes are unknown in function, as shown in Table 3.
TABLE 3 species-specific genes of Gelson Xinghua Nocardia, ningxia Nocardia and Yinnuoka
NA: due to differences in gene prediction software, the gene can be obtained from the reference genome location without alignment to the corresponding CDS sequence of the reference genome.
Primer design:
the specific genes are selected by comprehensively considering factors such as conservation in species, GC content and the like, and primers are designed by using Clone Manager v8 software and are evaluated to avoid forming primer dimers. After determining the conservation of the primers using MEGA v7 software, BLAST alignment was performed via NCBI, further confirming the specificity of the primers. Through experimental screening, the species-specific primer sequences of the Gerson-Xinghua bacteria, ningxia-nocardia and Yinchuan nocardia are finally obtained, and are shown in Table 4.
TABLE 4 species-specific primers for Gelson Xinghua Nordic acid bacteria, ningxia Nordic acid bacteria and Yinnuo Nordic acid bacteria
Example 1 construction of a complex population qPCR detection System of Gelson-Xinghai Nordic bacteria
1. Extracting genome: the genome of the strain was extracted using a genomic DNA purification kit. DNA purity and concentration were measured using a NanoDrop ND-1000 spectrophotometer. The genome DNA is packaged in small quantity and then is preserved at-20 ℃ for standby.
2. The reaction system: 12.5 Mu L2 XTB Greenpremix Ex Taq TM GC (Perfect Real Time), 0.5. Mu.L of forward and reverse primer (10. Mu.M), 1. Mu.L of DNA template (concentration approximately 100 ng), and 25. Mu.L of Dnase/Rnase-free deionized water.
3. Reaction conditions: qPCR was performed using a Roche real-time fluorescent quantitative PCR instrument. Pre-denaturation at 95℃for 1 min; denaturation at 95℃for 15 s, annealing at 68℃for 30 s,40 cycles; melting curves were obtained at 95℃15 s, 68℃1 min, and 95℃15 s.
4. Interpretation of the results: results analysis was performed using the LightCycler480 software. When the Ct value is less than or equal to 35, the detection result can be reported to be positive.
Example 2 evaluation of sensitivity of the composite population qPCR detection System of Gelson-Cruciferae
1. Preparation of a cationic quality control product:
and adding enzyme cutting sites on the upstream and downstream of the primer to amplify the target gene. 50. Mu.L of the reaction system comprises 25. Mu.L of Q5 super fidelity 2X Master Mix,2.5. Mu.L of forward and reverse primers (10. Mu.M), 2. Mu.L of target strain genome template and ultrapure water to 50. Mu.L. The PCR reaction conditions were determined and the annealing temperature was 68 ℃. After DNA electrophoresis using a 1.5% agarose gel and confirming that the band was single and the size was as expected, the PCR product was purified using a DNA purification kit. The PCR product and pUC57 plasmid were digested simultaneously at 37℃for 3 to 4 hours. After cleavage, purification was performed using a DNA purification kit. The purified PCR product and plasmid were mixed in a molar ratio of 1:4 and added to the Solution I reagent in an equal volume DNA ligation kit for enzymatic ligation at 16℃overnight. The ligation products were transformed into BL21 (DE 3) competent cells using chemical transformation. Plasmids were extracted according to the plasmid miniprep kit instructions.
2. Sensitivity of the detection system:
the recombinant plasmid containing the target fragment was 10-fold diluted with Dnase/Rnase-free deionized water to give a recombinant plasmid concentration gradient of 10 8 ~10 0 COPies/. Mu.L. qPCR amplification was performed by setting three parallel samples simultaneously with 10-fold serial dilutions of recombinant plasmid as template and Dnase/Rnase-free deionized water as blank. After amplification is completed, a standard curve is drawn with the logarithm of the initial DNA copy number and Ct value to determine the lower detection limit of the qPCR reaction. Typical S-shaped nucleic acid amplification curves (FIGS. 1, 4 and 7) appear in the detection results, and the amplification products have specific melting curves (FIGS. 2, 5 and 8).The analysis results of the standard curve data show (figures 3, 6 and 9) that the R of the qPCR method of the Gerson-Nakava, ningxia and Yinchun-Ka 2 Are all larger than 0.99, the amplification efficiency is between 90.25% and 96.15%, and the detection lower limit is 10 2 ~10 1 The orders of magnitude of copies/. Mu.L, and the relevant evaluation parameters are shown in Table 5. The amplification curve, standard curve and melting curve of the three nocardia qPCRs are shown in FIGS. 1-9.
TABLE 5 evaluation parameters of qPCR Standard curve
Example 3. Specificity evaluation of the complex population qPCR detection system of ganciclovir:
under standard reaction conditions, 40 strains of Gelson-Xinghuo Nocardia, six strains of Ningxia Nocardia and 15 strains of Yinchuan Nocardia DNA are used as templates, amplification curves can be generated, and the detection results of the three Nocardia have no cross reaction with the detection results of the traditional separation culture method and the whole genome identification sequencing result, so that the invention can effectively identify the three Nocardia.
By adopting the method described above, the method can be used, seven Nocardia (Nocardia melitensis, nocardia otorhinocina, nocardia neocinacae, nocardia asiatica, nocardia phlegm, nocardia brazilian and Nocardia abscess) or standard strains and 17 Nocardia near-edge strains (Mycobacterium tuberculosis, mycobacterium mirabilis, mycobacterium fortuitum, mycobacterium vaccae, mycobacterium vaccinum or their derivatives,M. conceptionense、M. syngnathidarumThe qPCR method specificity was evaluated for nucleic acids of seven clinically common respiratory pathogens (Klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, streptococcus pneumoniae, haemophilus influenzae, staphylococcus aureus and Moraxella catarrhalis). The results show that seven nocardia, 17 near non-target strains and seven clinical common respiratory tract pathogensNo specific amplification was generated and the qPCR method based on specific genes showed 100% specificity.
Example 4. Detection System applied to detection of clinical samples of human respiratory tract:
the nine nocardia positive human respiratory clinical samples (six sputum and three alveolar lavages) stored in this experiment were subjected to qPCR detection. The sample has been strain identified, isolated and cultured, and the genome is extracted and compared with the genome of the model strain for ANI. Each clinical sample was amplified with three pairs of primers, and the results are shown in Table 6, wherein 7 of the 9 samples were Gelson-Xinghuoka, 1 Ningxia-chu, and 1 Yinchuan-chu, and there was no cross reaction between the three pairs of primers.
TABLE 6 clinical sample test results
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Claims (7)

1. A primer combination for use in a polymerase chain amplification reaction, the combination comprising:
(1) The sequence of the upstream primer and the downstream primer of the polymerase chain amplification reaction designed by taking NCTC10797_05230 of Gelson's Nocardia is shown as SEQ ID NO.1 and SEQ ID NO.2 respectively;
(2) The IU407_RS05750 of Ningxia nocardia is used as a polymerase chain amplification reaction upstream primer and a downstream primer designed by target genes, and the sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4; and
(3) And the sequences of the upstream primer and the downstream primer of the polymerase chain amplification reaction designed by taking NOCYR_RS03685 of the Nocardia guarantor as target genes are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
2. The primer combination of claim 1, wherein the polymerase chain amplification reaction is a real-time fluorescent quantitative polymerase chain amplification reaction.
3. A kit comprising the primer combination of claim 1, wherein the kit is a chimeric fluorescent dye method real-time fluorescent quantitative polymerase chain amplification reaction.
4. The kit of claim 3, further comprisingTaqPolymerase, dntps, and fluorescent dye.
5. The kit of claim 3, wherein the fluorescent dye is TB Green.
6. Use of the primer combination of claim 1 for preparing a kit for detecting a complex population of ganciclovir bacteria.
7. The use according to claim 6, wherein the complex population of ganciclovir comprises ganciclovir, ganciclovir and ganciclovir.
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