CN117344039A - Primer and probe with high bacterial domain coverage rate, design method and application thereof - Google Patents
Primer and probe with high bacterial domain coverage rate, design method and application thereof Download PDFInfo
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Abstract
The invention provides a primer and a probe with high coverage rate of a bacterial domain, a design method and application thereof. Belongs to the field of sterile detection technology, nucleic acid amplification technology, cell products and microbial safety quality evaluation. The primer comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ_No.1, the sequence of the downstream primer is shown as SEQ_No.2, and the sequence of the probe is shown as SEQ_No. 3; the invention designs a group of universal qPCR primers and probes with high coverage rate and strong specificity for targeting the 16S rRNA genes, which are used for detecting bacterial pollution of cell products, and effectively improves the reliability and efficiency of the microbial safety quality evaluation technology of the cell products based on the nucleic acid amplification technology.
Description
Technical Field
The invention relates to a primer and a probe with high coverage rate in a bacterial domain, a design method and application thereof, and belongs to the fields of sterile detection technology, nucleic acid amplification technology, cell products and microbial safety quality evaluation.
Background
Compared with the traditional sterile biological products, the cell products have higher requirements on the sterile guarantee and process control of the production process and the quick release of finished products, and the pollution inspection of microorganisms is an important index for controlling the safety property of the cell products. Traditional aseptic testing methods rely on the natural growth of microorganisms on culture media, take a long time, require at least 14 days of culture to observe the growth of microorganisms [1], and cannot meet the process control and rapid release requirements of cell products at all.
With the rapid development of microbiology and molecular biology, the pharmaceutical field has introduced a variety of rapid microbiological detection methods, including the nucleic acid amplification techniques described in united states pharmacopeia code 1223, european pharmacopeia code 5.1.6, and chinese pharmacopeia code 9201. The fluorescent Quantitative PCR (qPCR) technology based on the probe has high sensitivity, strong specificity and short detection time, and is an effective means for rapidly detecting microorganisms on cell products.
The conserved region of the 16S rRNA gene sequence of the prokaryote is amplified by utilizing the qPCR technology of a probe method, so that bacterial pollution of a cell product is detected, and the method has been applied to process control of the cell product and rapid release of a finished product. However, the application history of the method in the aspect of medicine quality control is short, and the accumulated data in the aspect of broad spectrum detection is limited, so that the broad spectrum detection method needs to be fully evaluated before application.
Various universal primer and probe sequences targeting the prokaryotic 16S rRNA gene have been developed, and broad-spectrum property evaluation of methodology is generally confirmed by detecting representative species of various bacteria. However, the variety of bacteria is huge, and the variety of microorganisms involved in experimental verification is very limited. It cannot be demonstrated whether the 16S rRNA gene detection primer pairs considered universal actually cover the whole prokaryotic spectrum. For example, some evidence suggests that even the well-known primer pairs "27F" and "1492R" [2] for the highly conserved region of the 16S rRNA gene are not entirely universal [3,4]. At present, the universality of qPCR primers and probes for targeting the 16S rRNA gene has not been fully evaluated.
Therefore, there is a need in the art for qPCR primers and probes that target the bacterial domain of the 16S rRNA gene with high coverage to improve the broad spectrum of detection methods.
Disclosure of Invention
The invention aims to solve the problem of low coverage rate of qPCR primers and probes for targeting 16S rRNA genes in the prior art.
In order to solve the problems, the technical scheme adopted by the invention is to provide a primer and a probe with high coverage rate of a bacterial domain, and a design method and application thereof.
In a first aspect of the invention, a primer and a probe with high bacterial domain coverage rate are provided, wherein the primer comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ_No.1, the sequence of the downstream primer is shown as SEQ_No.2, and the sequence of the probe is shown as SEQ_No. 3;
SEQ_No.1：CCTACGGGWGGCWGCAG；
SEQ_No.2：GGACTACCRGGGTMTCTAATC；
SEQ_No.3：FAM-CAGCAGCCGCGGTA-MGB；
wherein W represents a degenerate base A/T, R represents a degenerate base A/G, and M represents a degenerate base A/C; FAM is a fluorescent group and MGB is a fluorescence quenching group.
In a second aspect of the present invention, there is provided a method for designing a primer and a probe with high coverage of a bacterial domain, comprising the steps of:
step 1: establishing an evaluation standard of the bacterial domain high coverage primer and the probe:
according to the existing literature and detection method, the coverage rate of the existing universal primers and probes on common microorganism groups in the bacterial domain is measured; wherein the common microorganism group in the bacterial domain comprises Proteus, actinomycetes, thick-walled bacteria and Bacteroides;
the coverage rate of the bacterial domain high coverage rate primer in the bacterial domain and the common microorganism group in the bacterial domain is over 78 percent, and the coverage rate of the bacterial domain high coverage rate probe in the bacterial domain and the common microorganism group in the bacterial domain is over 85 percent;
step 2: searching the 16S rRNA gene sequences of various bacteria in NCBI (National Center for Biotechnology Information), and carrying out homology analysis on the sequences by utilizing Clustal Omega to obtain a high-conservation region sequence of the bacteria;
step 3: designing a primer and a probe sequence to amplify a partial conservation region of the 16S rRNA gene according to the basic principle of qPCR primer and probe design [5 ];
step 4: calculating the annealing temperature of the primer by using an OligoCalc on-line calculator and evaluating the amplification performance of the primer and the probe;
step 5: evaluating the specificity of the primers by using the Primer-blast function of NCBI;
step 6: and (3) adopting the standard in the step (1), carrying out coverage rate assessment on the primers and probes designed in the step (3), the step (4) and the step (5), if the coverage rate is within the coverage rate range specified in the step (1), namely, the design is completed, otherwise, carrying out the step (3), the step (4) and the step (5) again, and carrying out coverage rate assessment again until the coverage rate falls into the coverage rate range specified in the step (1).
In a third aspect of the invention, there is provided an application of a primer and a probe with high coverage rate in bacterial domain in the sterile rapid detection of cell products, wherein the coverage rate of the used universal qPCR primer and probe is evaluated before the sterile rapid detection is carried out, and whether the primer and probe really have broad spectrum is judged.
Preferably, the primers and probes are the primers and probes with high coverage rate of the bacterial domain.
Compared with the prior art, the invention has the following beneficial effects:
the invention designs a group of universal qPCR primers and probes with high coverage rate and strong specificity for targeting the 16S rRNA genes, which are used for detecting bacterial pollution of cell products, and effectively improves the reliability and efficiency of the microbial safety quality evaluation technology of the cell products based on the nucleic acid amplification technology.
Drawings
FIG. 1 is a diagram showing the distribution of a primer and a probe of the present invention in a 16S rRNA gene;
FIG. 2 is a graph showing the evaluation of amplification performance of an upstream primer in the present invention;
FIG. 3 is a graph showing the evaluation of amplification performance of a downstream primer according to the present invention;
FIG. 4 is a graph showing the evaluation of the amplification performance of a probe according to the present invention;
in FIG. 1, the green modules represent 10 conserved regions C1-C10 of the 16S rRNA gene, and the blue modules represent 9 variable regions of the 16S rRNA gene. The upstream primer is located in a C3 conserved region of the E.coli 16S rRNA gene, the downstream primer is located in a C5 conserved region of the E.coli 16S rRNA gene, and the probe is located in a C4 conserved region of the E.coli 16S rRNA gene.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with the accompanying drawings are described in detail as follows:
comparative example 1 bacterial domain coverage was assessed for commonly used primers and probes.
Through literature retrieval and screening, the 16S rRNA qPCR primer and the probe sequence passing qualification standards are incorporated into a generality analysis to obtain coverage rate evaluation of the primer and the probe sequence on bacterial domains,
the document searching and screening steps are as follows:
step 1: document retrieval, in which the search field is set as all fields in an English online database, the used search words are bacterial qPCR, microbe qPCR, 16S rRNA qPCR, boolean operator AND, OR AND NOT are used for expanding AND shrinking the search range;
step 2: document screening, which is divided into title abstract screening (preliminary screening) and full text screening (rescreening);
the title abstract screening method comprises the following steps: importing authors, publishing time, titles and summaries of all documents retrieved in the document retrieval process into document management software, reading titles and summaries of the imported documents one by one, reserving documents which accord with the subject, namely comprise bacteria detection by using a probe method qPCR technology, and performing full text screening on the documents obtained by title summary screening;
the full text screening method comprises the following steps: downloading and reading the full text of the potential document obtained through title abstract screening and the complementary materials thereof, inquiring the primer and probe sequence of the targeted 16S rRNA gene, and evaluating and determining whether to incorporate analysis according to qualification standards;
step 3: qualification criteria screening:
3.1 only primer and probe sequences targeting the 16S rRNA gene were included in the study;
3.2 only the primers and probe sequences used in the technique of qPCR with the probe method are included in the study, and the primer pair used in qPCR with the dye method without the probe is not considered;
3.3 only broad-spectrum primers and probe sequences targeting the 16S rRNA gene were included in the study, irrespective of the species-specific primers and probe sequences of the bacterial domain;
3.4 only primer and probe sequences located between 45-1430 bp (Silva database alignment) of E.coli 16S rRNA gene sequences were investigated;
3.5 considering only primer sequences with amplified fragments smaller than 500 bp;
3.6 primers and probe sequences of the high primer were preferentially included in the study.
The coverage rate evaluation method of the bacterial domain comprises the following steps:
coverage rate evaluation is carried out on the primer and the probe sequence passing qualification standards by using a TestPrime module and a TestProbe module in a Silva database Search function respectively, wherein an SSU r138.1 is selected for the database, and a RefNR is selected for the type of the database; the complete match of the primer and probe sequences with the database sequences, i.e. "0mis match", is required to obtain the complete match coverage of the primer and probe sequences, and to study the classification of microorganisms involved in common bacterial contamination of cells, including the coverage of primers and probes in Proteus, actinomyces, thick-walled bacteria, bacteroides.
The results of coverage evaluation of the literature search and screening and bacterial domains are shown in table 1:
TABLE 1 coverage of bacterial domains by primer and probe sequences
Note that: 1. sequence position refers to the position of the primer and probe sequence in the E.coli 16S rRNA gene.
2. Coverage refers to the coverage of the upstream and downstream primers in the bacterial domain when the sequences match 100% of the 16S rRNA gene, wherein the data in brackets indicate the coverage of the probe sequences in the bacterial domain.
As can be seen from Table 1, the coverage of the widely used generic qPCR primers targeting the 16S rRNA gene was between 76.2% and 7.6% and the coverage of the probe was between 89.1% and 2.5%, indicating that the broad-spectrum bacterial domain primers and probes currently used did not completely cover all known bacterial taxa. Among them, the primer set designed by Corless CE et al [10] is often used as a general primer targeting the 16S rRNA gene, but its actual gene coverage is extremely low, less than 10%. This suggests that evaluating the versatility of the primers is far from being confirmed by detecting only a representative species of a partial bacterial taxa. In particular, in the aseptic detection of cell preparations, the bacterial domain coverage of the primers used in the nucleic acid detection technique directly affects the quality assessment and release of the cell preparations, and if the aseptic detection is performed without using the highly universal primers which are accurately assessed, serious consequences of release of the problematic preparations are very likely to occur.
In addition, although the coverage of the primer pair designed by Klascik S et al [6] is high, the coverage of the matched probe sequence pair 16S rRNA gene is extremely low, only 2.5%, which directly reduces the overall reliability of qPCR detection. This suggests that for the evaluation of qPCR detection efficiency, it is not feasible to explore only the coverage of the upstream and downstream primers, and detection of the probe coverage used must be matched to comprehensively evaluate the ability of qPCR technology to target the 16SrRNA gene.
Comparative example 2 evaluation of coverage of commonly used primers and probes on respective microbiota
Common bacterial contamination of cell products includes source contamination of raw materials and auxiliary materials and environmental pollution, wherein bacteria are mainly distributed in Proteus, actinomycetes, thick-walled bacteria and Bacteroides in a bacterial domain. The coverage of qPCR primer and probe sequences of the 16SrRNA gene by qualification criteria in the common microbiota was evaluated to obtain table 2.
TABLE 2 coverage of primer and probe sequences for common microbiota
Note that: coverage refers to the coverage of the upstream and downstream primers at each microbiota when the sequences match 100% of the 16S rRNA gene, wherein the data in brackets represent the coverage of the probe sequences at each microbiota.
As is clear from Table 2, the coverage of each primer pair and probe pair was not uniform for different microorganism taxonomies, for example, the coverage of primers designed by Yang S et al [9] was 50.8% for the phylum of Thick-walled bacteria, but only 0.4% for the phylum of actinomycetes. This would result in omission of actinomycete microorganisms in the detection of microorganisms. Therefore, in order to evaluate the versatility of the primer and probe, not only the coverage of the whole bacterial domain but also the coverage of the whole bacterial domain in each microorganism classification group should be considered. For rapid sterile detection of cell products, the coverage rate of the cell products in the common cell-contaminated microorganism taxonomic group should be evaluated, so that the cell-contaminated microorganisms can be detected more widely and uniformly, and the accuracy and the reliability of detection means are improved.
In general, the efficient and valuable cell preparation sterile detection scheme uses primers and probes which have high coverage rate and strong universality and can detect various common polluted bacterial classification groups more widely and uniformly.
Example 1, bacterial Domain high coverage primer and Probe design and amplification Performance evaluation
The sequences of the 16S rRNA genes of various bacteria are searched in NCBI, homology analysis is carried out on the sequences by utilizing Clustal Omega to obtain the sequences of the highly conserved regions of the 16S rRNA genes of the bacteria, and the sequences of the primers and the probes are designed and obtained based on the basic principles of primer and probe design (see Table 3).
TABLE 3 high coverage primers and probe sequences for bacterial domains
Note that: sequence position refers to the position of the primer and probe sequence in the E.coli 16S rRNA gene.
Wherein, to improve the broad spectrum of the primer, degenerate bases are specifically designed to enhance the coverage of the primer in combination with the sequence matching of the primer to each taxonomic group of microorganisms. As shown in FIG. 1, the designed upstream primer is positioned in the conserved region of the E.coli 16S rRNA gene C3, the downstream primer is positioned in the conserved region of the E.coli 16S rRNA gene C5, and the probe sequence is positioned in the conserved region of the E.coli 16S rRNA gene C4. The length of the amplified fragment of the primer pair is about 466bp. The annealing temperatures of the upstream primer and the downstream primer are similar and are close to 60 ℃. The annealing temperature of the probe is 5-10 ℃ higher than that of the primer, and the method accords with the general principle of qPCR primer design of a probe method. In addition, the OligoCalc on-line calculator shows that the potential hairpin structure and 3' -end complementation of the upstream primer and the downstream primer and the probe sequence are not existed (as shown in figures 2-4), and the primer amplification efficiency is ensured.
Example 2 bacterial Domain high coverage primers and Probe specificity and coverage assessment
The primer and probe sequences designed by the invention are subjected to homology evaluation with human genome, fungal genome and viral genome sequences by utilizing the primer-blast function of NCBI so as to detect the specificity of the designed primer and probe sequences. As is clear from Table 4, the primer set was unable to amplify the human gene, fungal gene and viral gene. The primer and the probe sequence designed by the invention have high specificity, are very suitable for bacterial detection in the sterile detection of cell products, and have important significance for improving the primer amplification efficiency and reducing false positive results.
TABLE 4 high coverage primers and probe specificity assessment for bacterial domains
The broad spectrum of primer and probe sequences targeting the 16S rRNA gene was also evaluated, and the coverage of the primer and probe sequences in the bacterial domain and the respective microbiota are shown in Table 5. The coverage rate of the designed primer and probe sequence to the bacterial domain is 78.5% and 92.8% respectively, and is obviously improved compared with the coverage rate of the general primer and probe shown in the table 1. And in each common microorganism classification group, the coverage rate of the primer and the probe is obviously improved on the whole, and each microorganism classification group can be detected more widely and uniformly. The primer has high broad spectrum on the whole bacterial domain, and has good targeting property on various taxonomic groups related to common microbial contamination of cells, and is a good universal cell product sterile rapid detection primer and probe.
TABLE 5 bacterial domain high coverage primers and probe coverage assessment
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that the present invention is not limited to the foregoing and various other changes, modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Reference to the literature
1. Li Gao, pan Wei, chen Xin, shao Hong, chen Gang rapid microbiological methods for detection of respiratory signals are used for the investigation of sterility of cell preparations pharmaceutical applications 2020,37 (21) in China, 2594-2598.
2.Weisburg WG,Barns SM,Pelletier DA,Lane DJ:16S ribosomal DNA amplification for phylogenetic study.J Bacteriol 1991,173(2):697-703.
3.Vergin KL,Urbach E,Stein JL,DeLong EF,Lanoil BD,Giovannoni SJ:Screening of a fosmid library of marine environmental genomic DNA fragmentsreveals four clones related to members of the order Planctomycetales.Appl EnvironMicrobiol 1998,64(8):3075-3078.
4.Derakshani M,Lukow T,Liesack W:Novel bacterial lineages at the(sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNAgenes from bulk soil and rice roots of flooded rice microcosms.Appl EnvironMicrobiol 2001,67(2):623-631.
5.Thornton B,Basu C:Rapid and simple method of qPCR primer design.Methods Mol Biol 2015,1275:173-179.
6.Klaschik S,Lehmann LE,Raadts A,Book M,Hoeft A,Stuber F:Real-timePCR for detection and differentiation of gram-positive and gram-negative bacteria.JClin Microbiol 2002,40(11):4304-4307.
7.Takai K,Horikoshi K:Rapid detection and quantification of members of thearchaeal community by quantitative PCR using fluorogenic probes.Appl EnvironMicrobiol 2000,66(11):5066-5072.
8.Nadkarni MA,Martin FE,Jacques NA,Hunter N:Determination of bacterialload by real-time PCR using a broad-range(universal)probe and primers set.Microbiology(Reading)2002,148(Pt 1):257-266.
9.Yang S,Lin S,Kelen GD,Quinn TC,Dick JD,Gaydos CA,Rothman RE:Quantitative multiprobe PCR assay for simultaneous detection and identification tospecies level of bacterial pathogens.J Clin Microbiol 2002,40(9):3449-3454.
10.Corless CE,Guiver M,Borrow R,Edwards-Jones V,Kaczmarski EB,Fox AJ:Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.J ClinMicrobiol 2000,38(5):1747-1752.
Claims (4)
1. A primer and a probe with high coverage rate of bacterial domains are characterized in that the primer comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ_No.1, the sequence of the downstream primer is shown as SEQ_No.2, and the sequence of the probe is shown as SEQ_No. 3.
2. A design method of a primer and a probe with high coverage rate of a bacterial domain is characterized by comprising the following steps:
step 1: establishing an evaluation standard of the bacterial domain high coverage primer and the probe:
according to the existing literature and detection method, determining coverage rate of the existing universal primers and probes on the bacterial domain and common microorganism taxonomies in the bacterial domain; wherein the common microorganism classification group in the bacterial domain comprises Proteus, actinomycetes, thick-walled bacteria and Bacteroides;
the coverage rate of the bacterial domain high coverage rate primer in the bacterial domain and the common microorganism taxon in the bacterial domain should reach more than 78%, and the coverage rate of the bacterial domain high coverage rate probe in the bacterial domain and the common microorganism taxon in the bacterial domain should reach more than 85%;
step 2: searching the 16S rRNA gene sequences of various bacteria in NCBI, and carrying out homology analysis on the sequences by utilizing Clustal Omega to obtain a high conservation region sequence of the bacteria;
step 3: designing a primer and a probe sequence to amplify a partial conservation region of the 16SrRNA gene according to the basic design principle of qPCR primers and probes;
step 4: calculating the annealing temperature of the primer by using an OligoCalc on-line calculator and evaluating the amplification performance of the primer and the probe;
step 5: evaluating the specificity of the primers by using the Primer-blast function of NCBI;
step 6: and (3) adopting the standard in the step (1), carrying out coverage rate evaluation on the primers and probes designed in the step (3), the step (4) and the step (5), if the coverage rate is within the coverage rate range specified in the step (1), namely finishing the design, otherwise, carrying out the step (3), the step (4) and the step (5) again, and carrying out coverage rate evaluation again until the coverage rate falls into the coverage rate range specified in the step (1).
3. The application of the primer and the probe with high coverage rate in the sterile rapid detection of the cell product is characterized in that the coverage rate of the universal qPCR primer and the probe is evaluated before the sterile rapid detection of the cell product is carried out, and whether the primer and the probe really have broad spectrum is judged.
4. The method of claim 3, wherein the primers and probes are used for detecting bacterial contamination in a cell preparation according to claim 1.
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