CN112080556A

CN112080556A - Method for performing multiple rapid sequencing on gonococcus drug-resistant gene

Info

Publication number: CN112080556A
Application number: CN202010809876.6A
Authority: CN
Inventors: 张驰; 彭俊平
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-12-15
Anticipated expiration: 2040-08-13
Also published as: CN112080556B

Abstract

The invention belongs to the technical field of molecular biology detection, relates to a method for performing multiple sequencing on drug-resistant genes of bacteria, and particularly relates to a method for performing rapid sequencing on drug-resistant genes of neisseria gonorrhoeae by using a nanopore sequencing technology. Meanwhile, the invention also relates to a kit for detecting the gonococcus drug resistance gene, and the kit contains the polynucleotides of SEQ ID NO.1-SEQ ID NO. 28.

Description

Method for performing multiple rapid sequencing on gonococcus drug-resistant gene

Technical Field

The invention belongs to the technical field of molecular biology detection, relates to a method for performing multiple sequencing on drug-resistant genes of bacteria, and particularly relates to a method for performing rapid sequencing on drug-resistant genes of neisseria gonorrhoeae by using a nanopore sequencing technology.

Background

Worldwide, gonorrhea remains one of the most important and common sexually transmitted diseases, and according to the statistics of the world health organization, 2016 number of new cases of gonorrhea reaches 8700 ten thousand worldwide. In recent ten years, with the development of social economy, the incidence of gonorrhea in China increases year by year, and the characteristics of large population density and large population mobility in China also create favorable conditions for the spread of gonorrhea. The spreading situation of gonorrhea imposes a serious public health burden on society. The gonorrhoeae (Neisseria gonorrhoeae, also known as gonococcus) is caused by infection, and reasonable and normative antibacterial treatment is the most effective means for curing diseases and inhibiting the spread of the diseases. However, as the resistance of gonococci to traditional antibiotics continues to increase and the sensitivity to cephalosporins decreases progressively, there is a trend towards gonococci becoming superbacteria without drug availability. Gonococcal drug resistance has become a serious public health problem at present, and gonococcal bacteria are also one of twelve drug-resistant bacteria which are highlighted by the world health organization.

To address this problem, sensitive antibiotics need to be selected for treatment based on the drug-resistant phenotype of gonococci, thereby slowing the rate of progression of their resistance variations. Traditionally, pathogenic bacteria in a patient sample need to be separated for drug sensitivity test for identifying the drug resistance phenotype of bacteria, the operation process is complicated, the period is long, the sensitivity is low, and timely and accurate information is difficult to provide for treatment. Compared with the prior art, the method has the advantages that the genotype of the drug resistance of the bacteria is analyzed through a molecular biology technology, so that the drug resistance phenotype is predicted, the problems can be well solved, and the method is a current research hotspot. However, unlike other bacteria, the drug resistance mechanism of gonococcus is very complex, and the drug resistance related sites are distributed in more than ten drug resistance genes and are in various forms such as Single Nucleotide Polymorphism (SNP), mosaic-like variation, plasmid acquisition and the like.

PCR-Sanger sequencing is the method mainly used for sequencing the drug-resistant genes and identifying drug-resistant sites, and although the method has high precision, one limitation is that the reaction weight is low, only one gene can be amplified and analyzed in a single reaction, and the sequence also needs to be corrected and intercepted manually after the sequencing reaction. As the drug resistance of the gonococcus involves more than ten genes, the comprehensive identification of the gonococcus undoubtedly requires a large amount of repeated work, so that the whole identification process is very complicated, time-consuming and labor-consuming. Another important limitation is that the method is based on the isolation of gonococcal strains, whereas in some gonorrheal patients the bacterial load of the infection is low and the strain cannot be isolated, thus resulting in a very limited use of the method.

In order to compensate for these defects, some newer molecular biology techniques, such as fluorescence quantitative PCR high resolution melting curve analysis, MassARRAY nucleic acid mass spectrometry, etc., are used to solve the problem, and although these techniques can simultaneously perform multi-site analysis, they are limited by the characteristics of the techniques themselves, and the results only indirectly suggest the existence of some drug-resistant sites represented as SNPs, and the complete sequence of the drug-resistant gene cannot be obtained. By simply applying these techniques, important sequence information is missed, and unknown or novel variations cannot be explored.

The MinION Nanopore sequencer, introduced by Oxford Nanopore Technologies (ONT), is representative of third generation sequencers and has the characteristics of long read length, portability, and real-time production data relative to second generation sequencers. At present, application of a MinION sequencer to infectious disease pathogen identification and drug resistance characteristic analysis is a research hotspot in the field, but the existing established methods are all based on the idea of metagenomics, namely unbiased sequencing of nucleic acid extracted from a sample. In clinical samples, the nucleic acid of a host has absolute advantages, so that only a very small amount of pathogen and drug-resistant gene related sequences can be screened in a sequencing result, and all drug-resistant genes and molecular typing genes are difficult to cover; meanwhile, the accuracy of the MinION sequencer is low, and if the analysis precision of a single base level is to be achieved, the coverage needs to be greatly increased. In view of these two requirements, sequencing methods based on a metagenomics strategy alone are not satisfactory.

Therefore, we envision a strategy of sequencing with multiplex PCR amplicons in this project, specifically, performing a PCR reaction to directly amplify all ten genes related to gonococcal drug resistance from a clinical sample, analyzing the amplified products with a MinION sequencer, and finally designing a bioinformatics analysis process to achieve the purpose of automatically obtaining the complete sequence of the target gene from the original sequencing data with only a few simple commands. The data yield of the existing MinION sequencing chip is calculated, and a single chip can sufficiently analyze 24 samples. In addition, by optimizing a sequencing library construction scheme, the experimental process can be greatly simplified, and the experimental time is shortened; finally, the new method is not limited by laboratory conditions and equipment and can be accomplished using a laptop computer. The advantages can better make up for the short plate in the existing gonococcus drug-resistant gene detection method.

The method can also be extrapolated to other clinically important pathogens as a standardized paradigm. For example, it is suitable for use with bacteria that grow slowly but have a large number of drug resistant genes, such as Mycobacterium tuberculosis; it can also be used for the drug-resistant gene detection of some pathogens which are difficult to culture, such as mycoplasma, chlamydia, virus, etc. In conclusion, the method has a plurality of potential application prospects, can be used as an important ring in a bacterial drug resistance laboratory detection system, and provides a new strategy and thought for guiding clinical reasonable use of antibiotics and exploring a pathogen drug resistance mechanism.

Disclosure of Invention

The invention aims to provide a method for sequencing drug-resistant genes of neisseria gonorrhoeae.

The invention establishes a set of gonococcal drug-resistant gene sequencing method by using a multiplex PCR amplicon sequencing strategy based on a MinION nanopore sequencer. The new method can be directly applied to clinical samples, namely more than ten genes related to the drug resistance of the gonococcus can be simultaneously sequenced in one experiment without carrying out isolated culture on the gonococcus. The whole experiment process is simple, the consumed time is short, and the experiment can be completed only by one notebook computer. The method can make up the limitation of the prior art, thereby being capable of providing timely evidence for clinically making a correct gonorrhea treatment scheme, and providing a new technical means for researching the drug-resistant evolution law of gonococcus and discovering new variation.

The detection method comprises the following steps:

step 1, obtaining a sample:

mainly collects the urethra, the genital tract, the rectal swab and the urine of the patient as the detection sample.

Step 2, DNA extraction:

DNA extraction technology is adopted to extract DNA in the sample.

Step 3, PCR amplification:

PCR products (amplicons) were obtained by multiplex PCR amplification.

Step 4, sequencing by a MinION nanopore sequencer:

the PCR products (amplicons) were sequenced using a MinION nanopore sequencer.

And 5, analyzing data:

by sequencing a sample or a strain and using a data analysis process, the complete sequence of more than ten drug-resistant genes of the gonococcus can be obtained, and further, the complete sequence can be used for identification of drug-resistant sites and exploration and discovery of new drug-resistant sites.

The sequencing of the amplicon adopted by the invention has the characteristic of high targeting, and can enable researchers to efficiently obtain the complete sequence of the drug-resistant gene, master the variation information of the drug-resistant site of bacteria and discover novel variation. Each reaction can simultaneously carry out multiplex analysis on more than ten amplified genes, can realize high coverage, can also provide highly targeted re-sequencing even in a region difficult to sequence, can not need to carry out bacterial culture on a clinical sample compared with methods such as whole genome sequencing and the like, and can reduce sequencing cost and turnaround time.

The detection method of the invention is characterized by comprising the following steps:

1) designing PCR reaction primer and determining PCR reaction condition

The invention selects genes related to drug resistance of penicillin, tetracycline, macrolides, cephalosporins, spectinomycin and the like, and designs multiple PCR primers (table 1 and table 2). In designing multiplex PCR primers, the amplification product must cover antibiotic resistance sites, while the annealing temperature of the primers and the length of the amplified fragments are required to be substantially identical, and the specificity of the primers must be considered, i.e., the binding capacity to other members of the genus nethertherium other than neisseria gonorrhoeae is minimized.

2) Establishing an experimental process of MinION library construction and sequencing reaction:

as a new generation of sequencing technology which is just emerging in recent years, nanopore sequencing is far from being applied to the second generation sequencing technology which is developed and mature. In particular, there is no official recommendation or available procedure for constructing libraries using amplicon fragments, and therefore, it is necessary to select an appropriate library construction and sequencing protocol based on the results of the preliminary experiments, and the factors to be considered in the selection are: precision of data output, complexity of the experiment and duration of the experiment.

3) Establishing a bioinformatics analysis process:

the original sequencing data is transformed and analyzed to obtain a target gene sequence, and the following bioinformatics steps are required: the raw sequencing data is formatted and the data is split according to the barcode. And (4) performing quality control on the data, and removing low-quality data. And comparing the filtered data, and classifying the sequencing reads (reads) according to the target genes according to the comparison result. And (3) mutually correcting reads belonging to the same gene amplicon to obtain the sequence of the gene in the sample to be detected. And writing a script program to realize the process.

4) And optimizing the established method.

There are two main aspects that need to be optimized, one of which is: the method uses a multiplex PCR technology, the reaction weight in the system is more than 10, optimization is not carried out, and the abundance difference of amplified fragments of each gene in a product is possibly too large, so that the data quantity of certain genes is too small, and the identification precision is influenced. Therefore, it is necessary to adjust the concentration of each pair of primers to ensure that the amplification efficiency is substantially consistent. The second step is as follows: and determining the minimum data quantity required on the premise of ensuring the analysis precision.

Preferably, the detection method of the present invention comprises the following steps:

step 1, obtaining a sample;

mainly collecting the urine, the urine and the swab of the urethra, the genital tract and the rectum of a patient as detection samples;

step 2, DNA extraction;

extracting DNA in a sample by adopting a DNA extraction technology;

step 3, PCR amplification

1) Multiplex PCR reaction system

The method can be applied to DNA of various clinical samples (swabs and urine) and DNA extracted by isolated strains, but different PCR systems (table 3) and reaction conditions (table 4) are required to be used when the DNA of the clinical samples and the strains is amplified.

Since some non-target bacteria may be present in the clinical sample, it also carries a plasmid containing the beta Lactamase (beta-Lactamase) and tetM genes. Primers that amplify both genes are therefore not recommended for use in amplifying clinical samples. In addition, since the DNA content of the target bacteria in clinical specimens is much lower than that of isolated strains, different PCR systems are required for amplifying DNA of clinical specimens and strains.

2) Concentration of each primer in the primer pool

Through multiple preliminary experiments, we optimized the amount of primers added to amplify different genes in the primer pool (table 5). Through optimization, the PCR amplification efficiency of each gene can be basically consistent.

Step 4, sequencing by MinION nanopore sequencer

The products after the multiple PCR reaction are firstly subjected to nucleic acid quantification, if a plurality of samples need to be sequenced simultaneously, the same amount of PCR products are prepared for library construction for each sample according to the quantification result, and the total mass of the sample loading of each MinION chip is 700-800 ng. Each sample is then concatenated with a barcode fragment for subsequent bioinformatics procedures to distinguish the samples. After this step is completed, the sample is mixed and then purified using magnetic beads, completing the construction of the sequencing library. Finally, the prepared library is loaded on a sequencing chip, and sequencing is started.

Step 5, data analysis

A certain amount of sequencing raw data is collected according to the number of samples sequenced. The sequence of the gene of interest can be obtained by analysis using the procedure and software shown in FIG. 1.

Another object of the present invention is to provide a kit for sequencing drug-resistant genes of gonococci.

The kit of the invention comprises the polynucleotides of SEQ ID NO.1-SEQ ID NO.28,

wherein SEQ ID NO.1-SEQ ID NO.28 are forward and reverse primer sequences;

wherein, the drug resistance gene comprises: penA, mtrR, ponA, gyrA, parC, porB,23S rRNA,16S rRNA, folP, rpsE, rpsJ, blaTEM, tetM, and the strain identification gene is porA.

The PCR primer sequences of 13 drug-resistant genes and 1 strain identification gene are shown as follows:

reagents useful for laboratory procedures such as: solvents, buffer solvents, auxiliary materials, and the like.

The kit can comprise the reagent prepared from the components, the preparation method of the reagent is conventional technology, and the reagent only needs to uniformly mix various raw materials at normal temperature without special equipment and conditions.

The kit can be used for respectively containing different reagents and then packaging the reagents and the reagents in the same packaging box together, and the operation is carried out according to the method described in the specification when the kit is used.

Compared with the existing detection method, the detection method provided by the invention has the following beneficial effects:

at present, PCR-Sanger sequencing is a method for sequencing drug-resistant genes of gonococcus and mainly used for identifying drug-resistant sites, although the method has high precision, one limitation is that the reaction weight is low, only one gene can be amplified and analyzed in a single reaction, and after the sequencing reaction, the sequence also needs to be corrected and intercepted manually. As the drug resistance of the gonococcus involves more than ten genes, the comprehensive identification of the gonococcus undoubtedly requires a large amount of repeated work, so that the whole identification process is very complicated, time-consuming and labor-consuming. Another important limitation is that the method is based on the isolation of gonococcal strains, whereas in some gonorrheal patients the bacterial load of the infection is low and the strain cannot be isolated, thus resulting in a very limited use of the method.

In addition to the above-mentioned classical PCR-Sanger sequencing method, some newer molecular biology techniques, such as fluorescence quantitative PCR high resolution melting curve analysis, MassARRAY nucleic acid mass spectrometry, etc., are applied to solve this problem, these techniques can simultaneously perform multi-site analysis, but are limited by the characteristics of the techniques themselves, and the results only indirectly suggest that a part of drug-resistant sites represented as SNPs exist or not, and the complete sequence of drug-resistant genes cannot be obtained. By simply applying these techniques, important sequence information is missed, and unknown or novel variations cannot be explored.

The invention uses the strategy of multiplex PCR amplicon sequencing, can directly amplify more than ten genes related to gonococcus drug resistance from clinical sample nucleic acid by only one PCR reaction without separating and culturing bacteria in the sample, then uses a MinION sequencer to analyze the amplified product, and realizes the purpose of automatically obtaining the complete sequence of the target gene from the original sequencing data by only using a few simple commands through designing the bioinformatics analysis flow.

The invention is helpful to quickly and conveniently obtain the complete sequence of more than ten drug-resistant genes of gonococcus. In addition, the method can be extrapolated to other clinically important pathogens as a standardized paradigm. For example, it is suitable for use with bacteria that grow slowly but have a large number of drug resistant genes, such as Mycobacterium tuberculosis; it can also be used for the drug-resistant gene detection of some pathogens which are difficult to culture, such as mycoplasma, chlamydia, virus, etc. Has a plurality of potential application prospects

For the related terms, the following explanations and explanations are made:

PCR	polymerase chain reaction
		Sanger sequencing	Dideoxy chain termination sequencing, also known as first generation sequencing
read	I.e., sequencing read length, is the length of sequence that can be determined by a sequencing reaction

Drawings

FIG. 1: data analysis process and software used in each link

Detailed Description

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto.

Example 1 detection method of the invention

Please check whether the following can clearly express the detection process, and describe the standard that the person skilled in the art can implement the detection process according to the following method.

1) Primer design

We selected 14 genes of interest for amplification: includes 13 drug resistance related genes (penA, mtrR, ponA, gyrA, parC, porB,23S rRNA,16S rRNA, folP, rpsE, rpsJ, blaTEM, tetM) and 1 strain identification gene (porA). Considering that most of the drug-resistant genes are within 2000bp in length, and in order to shorten the PCR time, the amplification length of the PCR is selected to be about 2000bp, and for five genes (ponA, gyrA, parC,23S rRNA, tetM) with the length being more than 2000bp, the method can obtain a 1500bp sequence which comprises all drug-resistant sites; except for these five genes, the full-length sequences of the remaining genes can be obtained. The primer sequences and the obtained sequence lengths are shown in Table 2.

2) Multiplex PCR reaction system

The method can be applied to DNA of various clinical samples (swabs and urine) and DNA extracted by isolated strains. Since some non-target bacteria may be present in the clinical sample, it also carries a plasmid containing the beta Lactamase (beta-Lactamase) and tetM genes. Primers that amplify both genes are therefore not recommended for use in amplifying clinical samples. In addition, since the DNA content of the target bacteria in the clinical specimen is much lower than that of the isolated strain, it is necessary to use different PCR systems (Table 3) and reaction conditions (Table 4) when amplifying the DNA of the clinical specimen and the strain.

3) Optimization of each primer concentration in primer pool

4) Library construction and on-machine sequencing

5) Data analysis

Test example 1 test effects of the present invention

1. Accuracy of the method

1) Accuracy in evaluating the sequenced strains: we first detected 9 reference strains of gonococcus using the method established in the present invention, and for these 9 reference strains, we previously sequenced 13 target genes involved in our method using PCR-Sanger sequencing to obtain their accurate sequences, and used this as a standard sequence for evaluating the accuracy of the method. Then, the sequence obtained by sequencing by the method of the invention is compared with a standard sequence, and the comparison result shows that the accuracy of the sequence obtained by the method of the invention can reach more than 99.5 percent.

2) Accuracy in sequencing clinical samples was evaluated: we selected 11 clinical samples and their corresponding isolates, and used 13 target gene sequences obtained by PCR-Sanger sequencing of the isolates as standard sequences. Then 11 clinical samples are sequenced by the method, the obtained sequence is compared with a standard sequence, and the result shows that the accuracy obtained by the method can also reach 99.5 percent when the clinical samples are sequenced.

2. Sensitivity of the method

By adding different concentrations of strain DNA into negative clinical samples as background, preparing simulated infection samples with gradient concentrations, and detecting the simulated infection samples with the gradient concentrations, we obtain the method of the invention, and the sensitivity can reach 31 copies per reaction. The sensitivity can meet clinical requirements.

Test example 2:

compared with the existing method for detecting neisseria gonorrhoeae drug-resistant sites, the method has the following advantages:

Claims

1. a kit for detecting a gonococcus drug-resistant gene is characterized in that the kit comprises polynucleotides of SEQ ID NO.1-SEQ ID NO. 28.

2. The kit of claim 1, wherein the drug resistance gene comprises: penA, mtrR, ponA, gyrA, parC, porB,23S rRNA,16S rRNA, folP, rpsE, rpsJ, blaTEM, tetM, and the strain identification gene is porA.

3. Use of the kit of claim 1 for detecting a mutation in a drug-resistant site of neisseria gonorrhoeae.

4. Use according to claim 3,

step 1, obtaining a sample:

mainly collects the urethra, the genital tract, the rectal swab and the urine of a patient as detection samples,

step 2, DNA extraction:

extracting DNA in the sample by adopting a DNA extraction technology,

step 3, PCR amplification:

PCR products (amplicons) are obtained by multiplex PCR amplification,

step 4, sequencing by a MinION nanopore sequencer:

sequencing the PCR product (amplicon) with a MinION nanopore sequencer,

and 5, analyzing data:

5. Use according to claim 3,

step 1, obtaining a sample;

step 2, DNA extraction;

extracting DNA in a sample by adopting a DNA extraction technology;

step 3, PCR amplification

1) Multiplex PCR reaction system

The method can be applied to various types of clinical sample DNA or DNA extracted by separating strains, but different PCR systems and reaction conditions are required to be used when the DNA of the clinical sample and the strain is amplified, and the method comprises the following steps:

2) concentration of each primer in the primer pool

Step 4, sequencing by MinION nanopore sequencer

Firstly carrying out nucleic acid quantification on products after the multiple PCR reaction is finished, if a plurality of samples need to be sequenced simultaneously, preparing PCR products with the same quantity for library construction by each sample according to the quantification result, wherein the total mass of the sample loading of each MinION chip is 700-800ng, then connecting each sample with a bar code fragment for distinguishing the samples in the subsequent bioinformatics flow, mixing the samples after the step is finished, then purifying by using magnetic beads to finish the construction of a sequencing library, finally loading the prepared library on a sequencing chip and starting sequencing,

step 5, data analysis

And collecting a certain amount of sequencing original data according to the number of the sequenced samples, and analyzing by using software to obtain the sequence of the target gene.

6. A method for rapid sequencing of drug resistance genes of neisseria gonorrhoeae, for non-diagnostic purposes, comprising the steps of:

1) primer design

The target genes selected for amplification were 14: including 13 drug resistance-related genes (penA, mtrR, ponA, gyrA, parC, porB,23S rRNA,16S rRNA, folP, rpsE, rpsJ, blaTEM, tetM) and 1 strain identification gene (porA), the primer sequences and the obtained sequence lengths are shown in Table 2,

2) multiplex PCR reaction system

The method can be applied to DNA of various clinical samples (swabs and urine) and DNA extracted by an isolated strain, primers for amplifying beta-Lactamase (beta-Lactamase) and tetM genes are not recommended to be used for amplifying the clinical samples because some non-target bacteria possibly exist in the clinical samples and the plasmids also contain the beta-Lactamase and the tetM genes, and in addition, different PCR systems (table 3) and reaction conditions (table 4) are required to be used for amplifying the clinical samples and the DNA of the strain because the DNA content of the target bacteria in the clinical samples is far lower than that of the isolated strain.

3) Optimization of each primer concentration in primer pool

Through multiple preliminary experiments, the addition amount of primers for amplifying different genes in the primer pool is optimized (table 5), and through optimization, the PCR amplification efficiency of each gene can be basically consistent,

4) library construction and on-machine sequencing

5) data analysis

According to the number of the sequenced samples, a certain amount of sequencing raw data is collected and analyzed by using the flow and software shown in figure 1, and the sequence of the target gene can be obtained.