CN116445588A - Quantitative reference for rapid detection of pathogenic microorganisms and detection method - Google Patents

Quantitative reference for rapid detection of pathogenic microorganisms and detection method Download PDF

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CN116445588A
CN116445588A CN202310335680.1A CN202310335680A CN116445588A CN 116445588 A CN116445588 A CN 116445588A CN 202310335680 A CN202310335680 A CN 202310335680A CN 116445588 A CN116445588 A CN 116445588A
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pathogenic microorganisms
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quantitative reference
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sequencing
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张骥诚
王颖
廖瑞
方涛
龙志成
赵淑云
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Wuhan Easydiagnosis Biomedicine Co ltd
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Abstract

The invention discloses a quantitative reference for rapid detection of pathogenic microorganisms and a detection method, wherein the sequence of the quantitative reference provided by the invention is not overlapped with the nucleic acid sequence of any species, and the rapid detection method of pathogenic microorganisms is provided based on the quantitative reference. According to the invention, by introducing the quantitative reference, whether the library is successfully built or not can be intuitively judged through the sample concentration and the capillary electrophoresis result in the library building stage, so that the failure sample judging time and cost are greatly shortened, and the experimental period is further shortened; the method can be used for detecting the nucleic acid sample containing the human genome DNA, has low detection limit, can realize simultaneous detection of a large number of different types of pathogenic microorganisms, can shorten the detection time to 4-6h, and has important significance for rapid and instant detection of the pathogenic microorganisms.

Description

Quantitative reference for rapid detection of pathogenic microorganisms and detection method
Technical Field
The invention belongs to the technical field of pathogenic microorganism detection, and particularly relates to a quantitative reference for pathogenic microorganism detection and a detection method.
Background
The traditional detection of pathogenic microorganisms depends on in-vitro culture tests, and solid culture media or liquid culture media are mostly used for in-vitro culture, so that the detection period is longer because a certain time is required for the growth and propagation of the pathogenic microorganisms, and the growth speeds of different pathogenic microorganisms are very different, so that batch samples cannot be processed at the same time. In order to solve the problem, various automatic culture and identification systems are continuously generated, and the traditional identification method is gradually improved, so that the inspection speed is greatly increased. The tissue cell culture identification method is suitable for the pathogen living in the specialized living tissue cells, including viruses, rickettsiae, chlamydia and the like. The tissue cells sensitive to different pathogens are different, living cells are taken out from animal tissues sensitive to the pathogens and are subjected to primary culture in vitro or are subjected to subculture by using a pathogen sensitive cell line, and after the pathogens are inoculated into the corresponding tissue cells, the pathogens can be propagated and grown in the tissue cells, so that specific cytopathic effects are caused. The pathogen can also be directly inoculated into sensitive animals to cause specific pathological changes of corresponding tissues and organs. Often, pathogens can be identified based on these specific lesions. Serological and immunological tests are techniques for rapidly identifying pathogens by detecting antigens or antibodies to the pathogens through known antibodies or antigens, simplifying the identification procedure, and common methods include serum agglutination techniques, latex agglutination tests, fluorescent antibody detection techniques, synergistic agglutination tests, enzyme-linked immunoassay techniques, and the like. The application of the ELISA technology greatly improves the sensitivity and specificity of serological detection, and not only can detect pathogen antigens in samples, but also can detect antibody components of organisms. The conventional pathogenic microorganism detection method has the disadvantages of complex operation, long detection period and higher requirements on the technical level of operators. With the continued development of technology, the diagnosis of pathogenic microorganisms has been no longer limited to pathogen levels, but has been extended to gene levels. The nucleic acid sequences, i.e., gene fragments, of pathogenic microorganisms are specific, and detection of specific gene fragment sequences can be used to identify pathogenic microorganisms, as opposed to other non-pathogenic microorganisms. With the development of technology, gene detection technology is being vigorously developed, and becomes an important detection technology for detecting pathogenic microorganisms in clinical laboratories and basic laboratories.
Currently Sanger sequencing is a gold standard for sequencing and is also the most widely used sequencing technology, which can identify and detect different types of pathogenic bacteria. With the development of genomics technology, high-throughput sequencing technology (NGS) is increasingly and widely applied to various aspects of tracing, detecting, parting, drug resistance evaluation and the like of infectious diseases, and rapidly develops towards a rapid and economic direction. The metagenomic sequencing (mNGS) technology does not rely on sequence amplification of specific gene primers in clinical infection, but performs mixed sequencing on all DNA or RNA of a sample to be tested, and obtains classification information of pathogens by comparing sequencing data with a pathogen database, so that targeted detection of the sample can be completed in a short time, thousands of pathogens can be detected at a time, and detected pathogen types comprise viruses, bacteria, fungi, parasites and the like. The turn-around time of the second-generation sequencing-based detection varies from the receiving of the sample to the completion of data analysis according to the sequencing technology, the method and the bioinformatics analysis method, the average detection time is 48h, and in order to reduce the sequencing time, the current detection within 48h is that the single-ended sequencing is 50bp, and the sequencing belongs to the sequencing of a short sequence. The current cost of second generation sequencing detection is still obviously higher than that of the traditional laboratory detection method, and the high cost is one of the main factors which restrict the wide development of second generation sequencing in China. Moreover, the second generation sequencing platform is adopted to sequence the metagenome, and the platform has the problems of long sequencing time, short sequencing reading time, a great deal of human genome DNA pollution of a sequencing result, high sequencing data and difficult interpretation of the sequencing result.
The nano Kong Ba sequencing (Nanopore Targeted Sequencing) method is a representative gene detection technology, and the method is independent of traditional microorganism culture, and can rapidly and objectively detect suspected pathogenic microorganisms (including bacteria, fungi and viruses) in clinical samples. The detection project is based on a third generation single molecule sequencing (Nanopore) platform, and is a new generation single molecule real-time sequencing technology, which uses single molecule DNA (RNA) to sequence by presuming base composition through current change of a biological Nanopore. The main characteristics are that the reading length is longer, the average reading length can reach 20kb, and the maximum reading length can reach Mb level; by combining efficient genome nucleic acid extraction and library establishment, sequencing results are compared and analyzed with a professional medical microorganism database after bioinformatics processing and analysis, so that species information of all microorganisms in a sample and carried drug resistance and virulence gene information can be obtained. The method has wide detection range, can efficiently detect pathogenic microorganisms in various clinical samples such as venous blood, alveolar lavage fluid, cerebrospinal fluid, sputum and other body fluids and tissues, and is suitable for diagnosis of fever, difficult infection and acute critical diseases caused by unknown reasons.
Disclosure of Invention
The invention aims to provide a quantitative reference product capable of being used for rapidly detecting pathogenic microorganisms and a method for rapidly detecting the pathogenic microorganisms based on the quantitative reference product, and the method has the advantages of high success rate of library establishment, short experimental period, low detection limit and the like.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a quantitative reference for rapid detection of pathogenic microorganisms, which is synthesized artificially and whose sequence needs to be non-overlapping with the sequence of a gene of any species in an existing database; the non-overlapping is specifically: the sequence of the quantitative reference has a similarity of < 75% with any sequence of the same length in any species.
A rapid detection method of pathogenic microorganisms based on a Nanopore sequencing platform comprises the following steps of:
s1, placing a quantitative reference, a nucleic acid sample and a specific primer group in the same reaction system for multiplex PCR amplification, wherein the specific primer group contains a primer for specifically amplifying the quantitative reference;
s2, using the amplified product obtained in the step S1 as a template, carrying out tagged sequence amplification, connecting the amplified product with a sequencing joint, obtaining sequencing data by adopting a Nanopore sequencing platform, and analyzing the sequencing data to obtain pathogenic microorganism types contained in the sample to be detected.
In the method, 5-16 different quantitative reference substances can be added into each reaction system according to detection requirements, however, certain differences among the quantitative reference substance sequences are required to be kept, so that resolution based on the sequences is facilitated.
According to the invention, by adding the quantitative reference, whether the library is successfully built or not can be intuitively judged through the sample concentration and the capillary electrophoresis result in the library building stage, so that the judging time and cost of the failed sample are greatly shortened, the library can be reestablished by arranging the failed sample at the first time, and the experimental period is shortened.
In the method, the nucleic acid sample can contain the human genome DNA, namely, the human genome DNA is not required to be removed when the nucleic acid of the sample to be detected is extracted, so that the DNA and RNA extracted by using an automatic instrument such as a conventional magnetic bead method can meet the requirement of subsequent library establishment and sequencing.
The method provided by the invention is particularly suitable for clinical samples with extremely low pathogenic microorganism content, can finish detection when the total quantity of extracted DNA is more than 50pg, can remarkably improve the success rate of database construction of trace samples, and can realize normal detection when the copy number in a reaction system is as low as 2 copies and each reaction is carried out when a standard sample is used for testing. The clinical sample can be conventional blood, alveolar lavage, cerebrospinal fluid, hydrothorax and ascites, oral swab, pus and other types.
In addition, the method can ensure that the microorganism ratio in the sequencing data can reach more than 95% under the condition of not removing the human genome DNA after optimizing a multiple amplification system, thereby solving the problem of pollution of the human genome DNA in pathogenic microorganism detection.
In the method, the primers in the specific primer group are used for targeted amplification of target areas enriched with pathogenic microorganisms and quantitative references, and each pathogenic microorganism can be provided with a plurality of target areas (such as 5-10 target areas), so that the accuracy and stability of judging the pathogenic microorganism can be greatly improved.
Preferably, the invention can be used in a two-stage amplification mode, and the primers in the specific primer group used for the first round of amplification have the following characteristics: the primer consists of two parts, namely a general sequence positioned at the 5 'end and a specific sequence positioned at the 3' end, wherein the specific sequence is designed based on each target region. After the universal sequence is added to the 5' end of the specific sequence, the universal sequence is added to the amplified target strip in the first round of multiplex amplification, and the universal sequence is used as a primer binding site in the second round of amplification, so that serious amplification preference of different primers caused by the difference of amplification efficiency can be avoided, and sequencing Reads of a sample more accord with the actual distribution level.
Furthermore, in the method, the 5' end of the primer used for amplification in the step S2 is additionally added with phosphorylation modification, and the rapid amplification enzyme used during amplification can additionally add an A base at the extension end, so that the generated amplification product can directly carry out TA connection of a sequencing joint without carrying out additional end repair, A addition and purification, thereby greatly simplifying the library construction step and shortening the experimental time.
In the above method, the amplification product obtained in step S1 may be treated with T4 or T7 exonuclease, and then subjected to amplification of a tagging sequence after purification. The method comprises the following steps: and (3) after the multiplex PCR amplification in the step (S1) is finished, purifying, adding the purified product into a T4 or T7 exonuclease reaction solution, uniformly mixing, centrifuging, placing the mixture into a PCR instrument, incubating at 37 ℃ for 2-10 min, stopping the reaction, and purifying to obtain the amplification in the step (S2). During this treatment, T4 or T7 exonucleases digest incomplete paired DNA from non-specific amplification in multiple reactions.
In the method, by using a long-reading long Nanopore sequencing platform, amplified long fragment data can be directly used for detecting the whole fragment without splicing, and the whole fragment can be used for subsequent pathogenic microorganism data analysis.
In the method, pathogenic microorganism analysis software developed by Minde autonomy can be adopted to analyze data generated by sequencing, sequencing data can be directly analyzed when the sequencing data starts to be generated, experimental sequencing and data analysis are synchronously performed, pathogenic microorganism data generated by sequencing is monitored in real time, and an analysis report is synchronously completed when sequencing is completed.
The invention also provides a kit for rapidly detecting pathogenic microorganisms, which comprises a quantitative reference and a primer for specifically amplifying and changing the quantitative reference, wherein the sequence of the quantitative reference is not overlapped with the gene sequence of any species in a database, and the sequence length of the quantitative reference is 150 bp-2500 bp.
The beneficial effects of the invention are as follows:
(1) The invention provides a quantitative reference, which can intuitively judge whether the library is successful or not through sample concentration and capillary electrophoresis results in the library establishment stage by adding the reference and specific amplification primers thereof into a reaction system of multiplex PCR amplification, thereby greatly shortening the judging time and cost of failed samples, and can schedule the failed samples to be re-established into libraries at the first time and shorten the experimental period. Meanwhile, the quantitative reference can consume substrates and enzymes in the amplification reaction process, can ensure the stability of an amplification system, and can detect extremely low-abundance pathogenic microorganisms through exponential growth of the quantitative reference.
(2) According to the invention, a two-section amplification mode is adopted, on one hand, a general sequence is added to the 5' end of a specific primer designed based on a target area, and the general sequence is used as a primer binding site to access a tag sequence, so that serious amplification preference of different primers due to amplification efficiency difference is effectively avoided, sequencing Reads of a sample is more in accordance with actual distribution level, and detection of up to 100 pathogenic microorganisms can be realized at the same time; on the other hand, phosphorylation modification is additionally added at the 5' end of the second round of amplification primer, and A is additionally added at the extension end of the rapid amplification enzyme used in the amplification, so that the generated amplification product can be directly subjected to TA connection of a sequencing joint, the library construction step is greatly simplified, and the experimental time is shortened.
(3) The nucleic acid sample extraction in the invention does not need to remove the human genome DNA, and the DNA extracted by using conventional magnetic bead method and other automatic instruments can meet the requirement of subsequent library establishment and sequencing; is suitable for sample types with extremely low pathogenic microorganism content such as blood and the like, and can finish detection when the total amount of extracted DNA is more than 50 pg; meanwhile, when the standard sample is used for testing, normal detection can be realized when the copy number in the reaction system is as low as 2.
(4) Compared with the conventional mNGS detection technology, the method can finish detection of pathogenic microorganisms in 4-6 hours, and can realize rapid and instant detection in detection application of pathogenic microorganisms.
Drawings
FIG. 1 is a schematic flow chart of a method for rapidly detecting pathogenic microorganisms.
FIG. 2 is a graph comparing the results of Qsep100 assay of example 2 for different library samples.
FIG. 3 shows the results of sequencing data for different pooled samples in example 2.
FIG. 4 is a graph showing Qsep100 measurement results of different pooled samples in control group 1 in example 3.
FIG. 5 is a graph showing Qsep100 measurement results of different pooled samples in control group 2 according to example 3.
FIG. 6 shows the results of sequencing data for different pooled samples in example 3.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.
In the following examples, unless otherwise specified, the methods are conventional; the reagents and materials described, unless otherwise specified, are commercially available.
Example 1
The experimental flow principle of the rapid detection method of pathogenic microorganisms based on the Nanopore sequencing platform provided by the embodiment is shown in the figure 1, and specifically comprises the following steps:
(1) And extracting nucleic acid of the clinical sample to be detected.
The extraction method may be a method of simultaneously extracting DNA and RNA, and it is recommended to extract nucleic acid by a magnetic bead method using a Minde automatic extractor. Extraction method refers to the kit instructions and instrument instructions for use, and the extraction time is about 0.5h. The obtained nucleic acid sample can be directly subjected to multiplex PCR amplification without removing the human genome DNA.
(2) Multiplex PCR amplification of specific primers in the first round.
The primer used in the round of amplification consists of two parts, namely a universal sequence at the 5' end, wherein the sequence is introduced because the universal sequence is used as a primer binding site in the second round of amplification, so that the amplification preference caused by the excessively high amplification cycle number can be avoided; the second part is a specific sequence at the 3' end, which binds to a pathogenic microorganism region and can specifically enrich the target region to be sequenced. It is understood that the universal sequences are divided into the universal sequence F at the 5 'end of the upstream primer and the universal sequence R at the 5' end of the downstream primer.
The general formulation system for this round of amplification reaction is as follows:
mixing the prepared reaction system uniformly, performing instantaneous centrifugation, and performing amplification reaction on a PCR instrument:
this round of amplification takes about 90min.
Immediately after the reaction was completed, subsequent purification was performed. The amplified product was purified 1.3 x using conventional purification magnetic beads, and specific procedures are referred to the instructions for use of the purification magnetic beads.
(3) T7 exonuclease digestion.
Adding all purified products into eight rows of T7 exonuclease reaction liquid, wherein the T7 exonuclease reaction liquid is prepared by the following steps:
after mixing evenly and instantaneous centrifugation, the PCR reaction tube with finished sample is put into a PCR instrument for incubation for 5min at 37 ℃,2 mu L of stop solution is added and mixed evenly, and the subsequent purification is immediately carried out. The product was purified 1.3 x using conventional purification beads, for specific procedures reference the instructions for use of the purification beads.
(4) The second round of PCR amplification of the tagged sequence.
Directly using the PCR reaction product of the first round as a template, adding the following prepared reaction reagent, and carrying out the second round of tagged sequence amplification, wherein the preparation system is as follows:
mixing the prepared reaction system uniformly, performing instantaneous centrifugation, and performing amplification reaction on a PCR instrument:
the second round of amplification takes about 40min.
The tagged primer used in this step is phosphorylated at the 5' end, and the tagged primer includes two parts, namely, a region corresponding to the universal sequence and a tag sequence region. The F end and the R end of the primer form a pair, each sample is amplified by using a pair of labeled primers, different samples use different labeled primers, and the different samples can be split directly based on the labeled primers during sequencing. The structure of the primers and tagged primers in the specific primer set with the universal sequence can be referred to patent CN 114196743A.
(5) Ligation of sequencing adaptors was performed.
The second round of amplification products were 1 x purified using conventional purification magnetic beads, and specific procedures are referred to the instructions for use of the purification magnetic beads. After purification, the Qubit concentration is measured, the samples to be sequenced in the same batch are mixed in equal quantity, and the total amount after mixing is 50-500 ng, so that the subsequent connection reaction can be performed.
The preparation of the connection system is as follows:
and (3) uniformly mixing the prepared reaction system, performing instantaneous centrifugation, connecting and reacting for 10min at 20 ℃, and connecting a sequencing joint after the reaction is finished. The ligation products were subjected to an on-machine reaction according to the sequence scheme of the Nanopore authorities. The ligation took about 0.5h to react with the machine.
(6) Sequencing and data analysis.
The data generated by sequencing are synchronously analyzed on Minde pathogenic microorganism analysis software, the sequencing time reaches 1-2 hours, the main pathogenic microorganisms can be detected, and the specific analysis flow refers to the software using instruction.
Example 2
The test method takes staphylococcus aureus standard as a sample to be tested, and the experimental process specifically comprises the following steps:
diluting an absolute quantitative staphylococcus aureus standard substance to 100 copies per reaction by a digital PCR instrument, extracting nucleic acid by a blood sample, adding 200ng of human DNA per reaction as a matrix, adding no quantitative reference substance into a control group sample, and adding 1 mu L of quantitative reference substance into an experimental group sample. The sequence of the quantitative reference product used in the example is shown as SEQ ID NO.1, and the specific sequence of the primer used is shown as SEQ ID NO. 2-5.
Library construction and sequencing were performed according to the procedure of example 1, and 2 samples were all normally subjected to library construction and sequencing data were obtained, and specific detection results were as follows:
and according to the concentration result of the Qubit determination, the sample of a control group without the quantitative reference substance is smaller than 1 ng/. Mu.L, and the sample of an experimental group with the quantitative reference substance is larger than 1 ng/. Mu.L.
The Qsep100 fragment size is shown in FIG. 2, a control sample without quantitative reference basically has no obvious peak diagram (left diagram), only has weak signal at 249bp, and an experimental sample with quantitative reference has obvious peak at 200-300bp (right diagram). If no quantitative reference is added, whether the library is successfully constructed is difficult to judge according to the concentration measurement result and the Qsep100 result; after the quantitative reference is added, whether the library is successfully constructed can be easily judged according to the concentration measurement result and the Qsep100 result.
The final sequencing data results show that, although the control sample without the quantitative reference can be detected at 20 copies/reaction, the number of sequences is only 1/10 of the experimental sample 2 with the quantitative reference, presumably because the PCR product concentration of the control sample is too low and too much loss occurs in the subsequent purification and library building steps.
Example 3
The test method takes staphylococcus aureus samples with different copy numbers as samples to be tested, and the experimental process specifically comprises the following steps:
taking an absolute quantitative staphylococcus aureus standard substance which is subjected to digital PCR (polymerase chain reaction) instrument, respectively diluting 100 copies of the standard substance to 2 copies of the standard substance, taking a blood sample, extracting nucleic acid, adding 200ng of human DNA (deoxyribonucleic acid) in a control group 1 as a substrate, adding 1 mu L of quantitative reference substance in each sample in a control group 2 without the substrate, carrying out library construction and sequencing according to the process of the invention (the quantitative reference substance sequence and the primer sequence are the same as those in the embodiment 2), and normally constructing libraries of 24 control samples and obtaining sequencing data, wherein the specific detection results are shown in the following table:
and according to the concentration result of the Qubit measurement, 24 samples are larger than 1 ng/. Mu.L, and the success of library construction can be primarily judged from the concentration. Qsep100 measures fragment size (FIGS. 4 and 5), and all samples had distinct peaks at 200-300bp, indicating successful library construction.
In 24 samples tested, 121 and 90 reads were obtained at as low as 2 copies per reaction, indicating that the lower detection limit of the kit was normally detectable at 2 copies per reaction. The control group 1 and the control group 2 can normally detect 90-6917 reads, which indicates that the product is not only suitable for samples with unmanned DNA as a matrix, such as cerebrospinal fluid and alveolar lavage fluid samples, but also can normally detect pathogenic bacteria in other samples with a large amount of human DNA, such as blood stream infection and the like.
In conclusion, through the use of the quantitative reference, whether the database is successfully built or not can be intuitively judged through the sample concentration and the capillary electrophoresis result in the database building stage, so that the time and the cost for judging the failed samples are greatly shortened; the detection method constructed by the invention can be used for detecting the nucleic acid sample containing the human genome DNA, has low detection limit, and can realize the simultaneous detection of a large number of pathogenic microorganisms of different types; and the detection time can be shortened to 4-6 hours, which has important significance for rapid and instant detection of pathogenic microorganisms.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims (10)

1. The quantitative reference for rapid detection of pathogenic microorganisms is characterized in that the sequence of the quantitative reference is not overlapped with the nucleic acid sequence of any species, and the sequence length of the quantitative reference is 150-2500 bp.
2. The rapid detection method of pathogenic microorganisms based on the Nanopore sequencing platform is characterized by comprising the following steps of:
s1, placing the quantitative reference substance, the nucleic acid sample and the specific primer group in the same reaction system for multiplex PCR amplification, wherein the specific primer group contains a primer for specifically amplifying the quantitative reference substance;
s2, using the amplified product obtained in the step S1 as a template, carrying out tagged sequence amplification, connecting the obtained amplified product with a sequencing joint, obtaining sequencing data by adopting a Nanopore, and analyzing the sequencing data.
3. The rapid detection method of pathogenic microorganisms according to claim 2, wherein the reaction system of step S1 contains 5 to 16 quantitative references of different sequences.
4. The method according to claim 2, wherein the nucleic acid sample in step S1 contains genomic DNA of human origin.
5. The method for rapid detection of pathogenic microorganisms according to claim 2, wherein the total amount of nucleic acids in the nucleic acid sample is not less than 50pg.
6. The rapid detection method of pathogenic microorganisms according to claim 2, wherein the nucleic acid sample is extracted from blood, alveolar lavage, cerebrospinal fluid, hydrothorax and ascites, oral swab or pus.
7. The method according to claim 2, wherein the primers in the specific primer set consist of a universal sequence at the 5 'end and a specific sequence at the 3' end.
8. The rapid detection method of pathogenic microorganisms according to claim 7, wherein the 5' end of the primer used for amplification in step S2 is modified by phosphorylation.
9. The method according to claim 2, wherein the amplification product obtained in step S1 is treated with T4 or T7 exonuclease, and the labeled sequence is amplified after purification.
10. The kit for rapidly detecting pathogenic microorganisms is characterized by comprising a quantitative reference and a primer for specifically amplifying the quantitative reference, wherein the sequence of the quantitative reference is not overlapped with any species, and the sequence length of the quantitative reference is 150-2500 bp.
CN202310335680.1A 2023-03-28 2023-03-28 Quantitative reference for rapid detection of pathogenic microorganisms and detection method Pending CN116445588A (en)

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