CN117210584A

CN117210584A - Kit for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids

Info

Publication number: CN117210584A
Application number: CN202310918959.2A
Authority: CN
Inventors: 阚兴池; 吴越; 曾梓菡; 黄柏成; 王鑫杰
Original assignee: Zhejiang Lab
Current assignee: Zhejiang Lab
Priority date: 2023-07-25
Filing date: 2023-07-25
Publication date: 2023-12-12

Abstract

The invention discloses a kit for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids, and relates to the technical field of biology. The excessive grinding selects the sputum sample as the detection object and has great clinical significance. The sputum in the deep part of the lung can accurately reflect the infection condition of pathogenic microorganisms in the lung tissue, and the sampling method belongs to noninvasive sampling, and can not cause relatively large stress reaction to patients. The detection method can detect streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae in sputum with high sensitivity and high specificity, so that the detection method has important clinical value. At present, a single pathogen detection method based on Cas12a is developed on the market, and the method can rapidly and accurately detect pathogen infection, but has narrow detection coverage range and relatively high detection cost, and the RPA/CRISPR detection method is beneficial to overcoming the defects.

Description

Kit for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids

Technical Field

The invention relates to the technical field of biology, in particular to a kit for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids.

Background

Respiratory diseases are serious hazards to human health, with pathogenic microbial infections, typified by bacteria, being a major factor in the initiation of respiratory diseases. Among them, four pathogenic microorganisms, streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae, are more common and more harmful. In the clinical diagnosis process, the sputum bacterial culture method (sputum culture) is a 'gold standard' for identifying pathogenic microorganisms, has strong clinical guidance significance and is an examination which is almost necessary for each respiratory disease patient. However, the experiment has the defects that the detection time is relatively long, the culture period of the pathogenic microorganisms in the sputum is about 5-7 days, so that the detection result of the sputum culture often cannot provide guidance for clinical treatment in time, and the detection rate of the pathogenic microorganisms in the sputum culture method is relatively low, so that the sputum culture method is an imperfect gold standard. Therefore, developing a new method for detecting the pathogenic microorganisms of the sputum is beneficial to enriching detection means of the pathogenic microorganisms of the respiratory tract and promoting the development of accurate medical treatment.

CRISPR-Cas is an adaptive immune system of bacteria, and Cas proteins can target cleavage of foreign nucleic acids under the guidance of RNA. The 2017-2018 team Zhang Feng and the Jennifer Doudna team report the side chain nonspecific efficient cleavage activity of CRISPR-Cas13a on single-stranded RNA viruses and CRISPR-Cas12a on double-stranded DNA viruses respectively, and the CRISPR-Cas system is applied to the field of nucleic acid detection, in particular to the field of disease specificity detection.

The current detection method based on CRISPR/Cas12a has great potential, and the detection method based on CRISPR/Cas12a is continuously developed in the diagnosis of infectious diseases. However, since different pathogenic microorganisms have large species differences, it is difficult to develop a CRISPR/Cas12a detection method in a standard manner, and it is necessary to optimize a detection system for different pathogenic microorganisms, such as reaction volume, pH, ion concentration, primer concentration, RPA primer concentration and ratio, cas12a concentration, crRNA concentration, cas12a to crRNA ratio, and the like.

Detection methods for single bacteria, such as a streptococcus pneumoniae home self-test kit based on a CRISPR auxiliary detection platform and a rapid and efficient detection method for mycoplasma pneumoniae based on an RPA-CRISPR/Cas12a technology, have been developed at present on the basis of CRISPR/Cas12 a. Although the above CRISPR/Cas12 a-based detection methods are sensitive and effective, only a single bacterial infection can be detected, whereas in clinical practice patients often are bacterial mixed infections. Obtaining the result of bacterial mixed infection using the above method requires the use of a large number of detection consumables and unnecessary human consumption. If multiple pathogen infection can be detected at the same time by one detection, the detection time and cost are greatly saved.

Streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae are four common pathogenic microorganisms that seriously jeopardize the health of the human respiratory system. In 2005, a method for detecting the four pathogenic bacteria based on a multiplex PCR technology was reported. PCR is a common method for nucleic acid detection, but the PCR detection method belongs to a first generation PCR method (qPCR is a second generation PCR method; digital PCR is a third generation PCR method) and can realize absolute quantification of nucleic acid molecules. The detection method needs to be carried out in a laboratory, is seriously dependent on expensive scientific research instruments, cannot generate visual fluorescence, and has long detection time, so that the detection method has a certain limitation in portable clinical application.

Disclosure of Invention

According to the defects of detecting pathogenic bacteria in clinical sputum culture at present, the invention develops a quadruple detection method of the pathogenic bacteria in sputum based on Cas12, and the invention has the advantages of no dependence on a large instrument, relatively simple and convenient operation, high detection specificity and sensitivity, relatively low detection cost, contribution to enriching technical means of detecting the pathogenic bacteria in sputum, and contribution to solving the problems of long detection period and low detection rate of sputum culture.

The specific technical scheme of the invention is as follows:

the invention provides a target for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids, which is characterized in that the target is a target combination consisting of four sequences, and the nucleotide sequence of the target combination is shown as SEQ ID NO. 21-24.

The invention also provides an RPA primer combination for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids, the primer combination comprising:

streptococcus pneumoniae (stp) specific primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2,

a haemophilus influenzae (Hai) specific primer with nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4,

a chlamydia pneumoniae (Chp) specific primer with nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO.6,

mycoplasma pneumoniae (Myp) specific primers with nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO. 8.

The invention also provides a crRNA combination for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids, the crRNA combination comprising:

streptococcus pneumoniae-specific crRNA having a sequence shown in SEQ ID NO.9,

haemophilus influenzae specific crRNA with the sequence shown as SEQ ID No.10,

chlamydia pneumoniae specific crRNA having the sequence shown in SEQ ID NO.11,

mycoplasma pneumoniae-specific crRNA having the sequence shown in SEQ ID NO. 12.

The invention also provides a full-length primer amplification primer for streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae plasmids, wherein the full-length PCR primer is as follows:

the upstream PCR sequence of the streptococcus pneumoniae detection target gene lytA is shown as SEQ ID NO.13 and the downstream PCR sequence SEQ ID NO.14,

the upstream PCR sequence of the haemophilus influenzae detection target gene lytA is shown as SEQ ID NO.15 and the downstream PCR sequence is shown as SEQ ID NO.16,

the nucleotide sequence of the chlamydia pneumoniae detection target gene Chp-ompa is shown as SEQ ID NO.17 and the downstream PCR sequence is shown as SEQ ID NO.18,

the nucleotide sequence of the mycoplasma pneumoniae detection target gene Myp-P1 is shown as SEQ ID NO.19 and a downstream PCR sequence SEQ ID NO. 20.

Preferably, the preparation method of the specific crRNA comprises: aiming at streptococcus pneumoniae lytA gene, haemophilus influenzae 16S-rRNA gene, chlamydia pneumoniae P1 gene and mycoplasma pneumoniae Ompa gene, a PAM (Protospacer adjacent motif) sequence TTTN is searched for, and crRNA is designed. After the design is completed, oligonucleotide (oligo) is synthesized by a chemical method to obtain target crRNA, and then the crRNA with optimal reactivity is obtained by Cas12a detection and used for method establishment and kit construction.

The invention also provides a kit for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acid, which comprises an isothermal amplification system, a detection system and a fluorescence detection system of an enzyme-labeled instrument;

the isothermal amplification system comprises the primer combination as isothermal amplification primer,

the Cas12a detection system includes the crRNA combination,

the microplate reader fluorescence detection system is used for detecting a Cas12a detection reaction product generated by the Cas12a detection system.

Specifically, the Cas12a detection system further includes Cas12a protein, ssDNA reporter system (ssDNA-FAM/BHQ 1, AZENTA) including ssDNA FQ reporter for fluorescent detection.

Wherein ssDNA FQ reporter is ssDNA labeled with 6-carboxyfluorescein (6-FAM) and fluorescence quencher (BHQ 1), and the labeling products are as follows: the ratio of the FAM/TTATT/3BHQ1/, which is named ssDNA FQ reporter/5.6FAM/TTATT/3 BHQ1/.

The kit provided by the invention can be used for fluorescence detection by using an enzyme-labeled instrument or irradiation by using a blue light gel cutting instrument. In fluorescence detection, the DNA (ssDNA) reporting system in Cas12a detection system is ssDNA FQ reporter.

Upon fluorescence detection, the Cas12a protein after activation of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae specific nucleic acids cleaves ssDNA FQ reporter labeled with a fluorescent group and a quencher group, thereby releasing the activating fluorescent group, and fluorescence reading can be detected using an enzyme-labeled instrument. Correspondingly, when no specific nucleic acid exists in the sample to be detected, no fluorescence reading exists.

The wavelength of the blue light gel cutting instrument is 470 nanometers, and a positive result of nucleic acid detection can be excited to generate a macroscopic fluorescent signal.

The invention also provides a method for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids, comprising the steps of:

(1) Extracting nucleic acid of a biological sample to be detected;

(2) RPA isothermal amplification and Cas12a detection: amplifying the biological sample nucleic acid obtained in the step (1) in an RPA isothermal amplification system by utilizing the primer combination, detecting the amplified product by using Cas12a, and reacting to obtain a Cas12a detection product;

(3) And performing enzyme-labeled instrument fluorescence detection or blue light gel cutting instrument irradiation on the Cas12a detection product.

In the step (1), the biological sample to be detected is a sputum sample.

In the step (2), the amplification condition is 39 ℃ for 20 minutes; the Cas12a detection conditions were 37 ℃ for 15 minutes.

Isothermal amplification-Cas 12a detection is carried out by adding a sample to be detected into an isothermal amplification reaction chamber, sucking isothermal amplification products into the Cas12a detection reaction chamber after reaction is completed, and finally judging detection results of all Cas12a detection reaction products through fluorescent signals to complete detection.

Specifically, the method for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids comprises the steps of:

s1: preparation of specific crrnas. Aiming at streptococcus pneumoniae lytA genes, haemophilus influenzae 16S-rRNA genes, chlamydia pneumoniae P1 genes and mycoplasma pneumoniae Ompa genes, designing crRNA comprising a Cas12a recognition sequence TTTN, constructing a vector, and performing in vitro transcription to obtain crRNA;

s2: nucleic acid release. Releasing nucleic acid in the sample to be tested by using a nucleic acid quick release reagent;

s3: isothermal nucleic acid amplification and Cas12a detection. The S2 step sample was amplified in the RPA system using isothermal amplification primers (reaction at 39 ℃ C. For 20 min), and the activated Cas12a cleavage probe (reaction at 37 ℃ C. For 15 min) was allowed to recognize nucleic acid sequences specific for Streptococcus pneumoniae lytA gene, haemophilus influenzae 16S-rRNA gene, chlamydia pneumoniae P1 gene or Mycoplasma pneumoniae Ompa gene by Cas12 a.

S4: the reaction product was irradiated with a blue light gel cutting instrument, and the detection result was interpreted by visual inspection.

The invention has the beneficial effects that:

1. the excessive grinding selects the sputum sample as the detection object and has great clinical significance. The sputum in the deep part of the lung can accurately reflect the infection condition of pathogenic microorganisms in the lung tissue, and the sampling method belongs to noninvasive sampling, and can not cause relatively large stress reaction to patients. However, the number of pathogenic bacteria contained in the sputum sample is relatively reduced, so that accurate detection has certain difficulty. But the detection method can detect streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae in sputum with high sensitivity and high specificity, so that the method has important clinical value.

2. At present, a single pathogen detection method based on Cas12a is developed on the market, and the method can rapidly and accurately detect pathogen infection, but has narrow detection coverage range and relatively high detection cost, and the RPA/CRISPR detection method is beneficial to overcoming the defects.

3. According to the detection method, four pathogenic microorganisms in a sputum sample can be detected with high sensitivity and high specificity by detecting the fluorescence condition of the non-specific side chain cleavage single-chain fluorescence reporter gene of the Cas12a protein.

Drawings

FIG. 1 is a schematic and flow chart of multiple detection of pathogenic microorganisms in a sputum sample.

FIG. 2 is a graph of detection sensitivity for Streptococcus pneumoniae; wherein p < 0.05.

FIG. 3 is a graph showing the detection sensitivity of Haemophilus influenzae; wherein p < 0.05.

FIG. 4 is a graph of detection sensitivity of Chlamydia pneumoniae; wherein p < 0.05.

FIG. 5 is a graph of mycoplasma pneumoniae detection sensitivity; wherein p < 0.05.

FIG. 6 is a graph of fluorescence results of a quadruple RPA/CRISPR method for detecting a plasmid sample nucleic acid target gene.

FIG. 7 is a graph of a statistical analysis of the RPA/CRISPR fluorescence detection results of FIG. 6; wherein p < 0.05.

FIG. 8 is a diagram showing the detection of pathogenic microorganism DNA in sputum samples from clinically positive patients.

FIG. 9 is a pUC57 plasmid map.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The general technical schematic diagram of the invention is shown in fig. 1, and comprises the following 4 parts: preparation of specific crRNA, nucleic acid release, isothermal amplification of nucleic acid and detection of Cas12a, and fluorescence detection by a blue light gel instrument.

The CRISPR/Cas12 a-based method for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids comprises the steps of:

Example 1

A kit and a method for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids:

1. preparation of specific crRNA

(1) Screening pathogenic bacteria to be detected in sputum

Through clinical investigation, a plurality of pathogenic microorganisms which have serious influence and are represented with a certain degree are selected as detection targets, and streptococcus pneumoniae (Streptococcus pneumoniae, stp), haemophilus influenzae (Haemophilus influenzae, hai), mycoplasma pneumoniae (Chlamydophila pneumoniae, chp) and chlamydia pneumoniae (Mycoplasma pneumoniae, myp) which are represented with a strong degree and are easy to cause infection of lung tissues in sputum are used.

(2) Synthetic pathogen detection plasmid

The detection genes of four pathogenic bacteria are synthesized by the biological engineering (Shanghai) stock company, and constructed on a pUC57 vector (map is shown in figure 9), and the full-length primer amplification primers for the plasmids of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae are shown in table 1.

(3) Synthesis and screening of specific crrnas

Within the scope of detection targets, 4-5 crrnas per gene are preferred depending on PAM site (TTTN). The crrnas were synthesized and tested in a matrix format for cross-reactivity of different crrnas with the test templates. The crRNA used in the project is a plasmid which has no cross reaction between the screened high-efficiency crRNA and different templates, results are shown in figure 6, and the results prove that multiple RPA products respectively generate fluorescence under the side chain cleavage of four pathogenic bacteria specific crRNAs, and the multiple RPA reaction system successfully amplifies four pathogenic microorganisms, including a lytA target gene fragment of streptococcus pneumoniae, a 16S-rRNA target gene fragment of haemophilus influenzae, a P1 target gene fragment of mycoplasma pneumoniae and an Ompa target gene fragment of chlamydia pneumoniae. The crrnas used in the present invention are therefore highly specific. Namely, the specific crRNA with the sequences shown in SEQ ID NO. 9-12 is obtained by corresponding to the sequence of the lytA target gene fragment of streptococcus pneumoniae, the 16S-rRNA target gene fragment sequence of haemophilus influenzae, the P1 target gene fragment sequence of mycoplasma pneumoniae and the Ompa target gene fragment sequence of chlamydia pneumoniae, and specific information is shown in Table 1. crRNA is responsible for synthesis by the company, inc.

2. Sample preparation

And releasing the nucleic acid in the sample to be tested by using the nucleic acid quick release reagent.

1-2g of sputum tissue was removed, sputum digestate (Sputasol method) was added, and about 5mL of concentrated sputum was lifted into a labeled 50mL centrifuge tube. Adding equal amount of sputum digestion liquid for homogenization treatment, vortex shaking for 20s, and standing for 15-20 min at 30-37 ℃. Sterile PBS (pH around 6.8) was added to about 50mL and centrifuged at 3000rpm for 15min. Pouring out the supernatant, adding 1-3 mL of PBS, and oscillating for 10min in a warm bath at 100 ℃ to obtain the nucleic acid in the sample to be detected.

3. RPA isothermal amplification and Cas12a detection

Pre-amplifying the pathogen nucleic acid using reverse transcription isothermal amplification (RT-RPA) to perform a Cas12a detection reaction;

according to the requirement of isothermal amplification reaction, 3 RPA primers are respectively designed at the upstream and downstream of the template according to the detection target in the step (2), the amplified products are controlled within 200bp, and the optimal primer combination mode is optimized according to the fluorescence signal intensity after the RPA primers are synthesized. The RPA primer used in the patent is a primer combination pair with the best effect after screening, and the sequence information is shown in Table 1. The RPA primer was synthesized by the division of biological engineering (Shanghai).

The research utilizes an enzymatic recombination isothermal amplification (Enzymatic Recombinase Amplification, ERA) technology, and the reaction system is 25 mu L, which specifically comprises: reaction buffer 10. Mu.L, ERA 5. Mu.L, 10. Mu.M upstream primer (SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7 in a 1:1:1:1 ratio by volume) 1.25. Mu.L, 10. Mu.M downstream primer (SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8 in a 1:1:1:1 ratio by volume) 1.25. Mu.L, agonist magnesium acetate (MgoAC, 350 mM) 1. Mu.L, DNA template 2. Mu.L (sample loading volume unchanged, different sample loading samples contain different concentrations of gene copy number), ddH ₂ O4. Mu.L, amplification conditions were 39℃for 20min. The RPA kit is purchased from suda gene technologies limited.

The CRISPR/Cas12a reaction system is 20 μl, comprising: ddH ₂ O8.9 mu L, RNA enzyme inhibitor 0.1 mu L, buffer 3.1.1 mu L, ssDNA reporter 1 mu L, cas a protein 1 mu L, crRNA (sequence shown in SEQ ID NO. 9-12) 2 mu L, RPA product 5 mu L. Wherein the ssDNA reporter system comprises ssDNA FQ reporter for fluorescence detection by an enzyme-labeled instrument.

In fluorescence detection, cas12a protein after activation of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae specific nucleic acids cleaves ssDNA FQ reporter labeled with a fluorescent group and a quencher group, thereby releasing the activating fluorescent group, and fluorescence can be detected using a blue-light glue instrument.

The nucleic acid of the sample to be detected is incubated for 20min at 39 ℃ through an isothermal amplification reaction system, then 5 mu L of isothermal amplification reaction product is reacted for 15min at 39 ℃ through a CRISPR/Cas12a reaction system, and the instrument for detecting the fluorescent signal of the reaction product used in the detection is a blue light gel instrument with the detection wavelength of 470-520nm.

FIG. 6 results demonstrate that multiple RPA products fluoresce upon side-chain cleavage of four species-specific crRNAs, respectively. The results of the research demonstrate that the multiplex RPA reaction system successfully amplified plasmids of four pathogenic microorganisms including Streptococcus pneumoniae (lytA), haemophilus influenzae (16S-rRNA), mycoplasma (P1) and Chlamydia (Ompa).

FIG. 7 is a statistical analysis of the results of FIG. 6, and FIG. 6 shows a detection system of the present invention, which contains CRISPR components and quadruple primers upstream and downstream of the detection. Sterile water was added to the negative control group and the samples added to the test group were different plasmids to be tested. Different groupings are distinguished by specific crrnas. The fluorescence intensities of the streptococcus pneumoniae, haemophilus influenzae, mycoplasma pneumoniae and chlamydia pneumoniae groups were all significantly increased compared to the control (no template control, NC) group. The result shows that the detection system for hyperthyroidism has scientificity and rationality, and a multiple detection method for streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acid is successfully constructed.

TABLE 1

Example 2 detection sensitivity of RPA/CRISPR

The DNA template prepared in example 1 was serially diluted to different gene copy numbers, including 1.0X10 ⁵ (also written as e 5), 1.0X10 ⁴ 、1.0×10 ³ 、1.0×10 ² 、1.0×10 ¹ And 0, respectively judging the detection sensitivity of the four pathogenic bacteria according to the fluorescence result. The detection results are shown in figures 2-5, and the results in figure 2 prove that the detection method has high sensitivity, and the detection sensitivity for streptococcus pneumoniae can reach 10 copies/. Mu.L; the result of FIG. 3 shows that the detection method has high sensitivity, and the detection sensitivity for haemophilus influenzae can reach 10 copies/. Mu.L; the results of FIG. 4 demonstrate that the detection method has high sensitivity, and the detection sensitivity for Chlamydia pneumoniae can reach 10 copies/. Mu.L; the results in FIG. 5 demonstrate that the detection method has high sensitivity, and the detection sensitivity for mycoplasma pneumoniae can reach 10 copies/. Mu.L.

Example 3 clinical testing

And detecting pathogenic microorganism DNA of sputum samples of clinically positive patients. In clinical practice, although single bacteria infect the respiratory tract, it is common, but there are many cases where multiple bacterial mixed infections exist. In view of this objective fact, this example randomly selected a positive patient who had been diagnosed by a doctor to have simultaneous infection with Streptococcus pneumoniae, haemophilus influenzae, mycoplasma pneumoniae and Chlamydia pneumoniae.

1. Preparation of samples

Sputum samples from clinically positive patients were randomly taken, from Hangzhou first-person hospitals, sputum digests (Sputasol method) were added and approximately 5mL of concentrated sputum was picked up into a labeled 50mL centrifuge tube. Adding equal amount of sputum digestion liquid for homogenization treatment, vortex shaking for 20s, and standing for 15-20 min at 30-37 ℃. Sterile PBS (pH around 6.8) was added to about 50mL and centrifuged at 3000r/min for 15min. Pouring out the supernatant, adding 1-3 mL of PBS, and oscillating for 10min in a warm bath at 100 ℃ to obtain a treated sample. 5 μl of the treated samples were taken for RPA amplification and CRISPR/Cas12a detection.

2. RPA isothermal amplification and Cas12a detection

And 5 mu L of the sample treated in the step of 1 and sample preparation is added into an isothermal amplification reaction system, and the mixture is incubated at 39 ℃ for 10 minutes. Then, by sucking 5 mu L of isothermal amplification reaction products into a CRISPR/Cas12a reaction system, reacting for 15min at 39 ℃ to obtain Cas12a reaction products; wherein the isothermal amplification reaction system and the CRISPR/Cas12a reaction system are the isothermal amplification reaction system and the CRISPR/Cas12a reaction system of step 3 in example 1.

3. Blue light glue instrument fluorescence detection

The Cas12a reaction product in the step 2 is used for determination by a blue-light glue analyzer, and the research result shows that the RPA/Cas12a detection method developed in the project successfully detects pathogenic microorganisms in sputum samples, including streptococcus pneumoniae, haemophilus influenzae, mycoplasma pneumoniae and chlamydia pneumoniae.

In conclusion, the experiment proves that the detection method has high sensitivity and portability, can rapidly detect four pathogenic microorganisms in sputum samples, enriches detection means of respiratory pathogenic microorganisms, and has important significance for guiding diagnosis of clinical respiratory pathogenic microorganisms.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The target for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids is characterized in that the target is a target combination consisting of four sequences, and the nucleotide sequence of the target combination is shown as SEQ ID NO.21 to SEQ ID NO. 24.

2. A primer combination for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids, the primer combination comprising:

streptococcus pneumoniae specific primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2,

the nucleotide sequences are shown as SEQ ID NO.3 and SEQ ID NO.4,

a chlamydia pneumoniae specific primer with nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO.6,

the nucleotide sequences are shown as SEQ ID NO.7 and SEQ ID NO.8 of mycoplasma pneumoniae specific primers.

3. A crRNA combination for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids, the crRNA combination comprising:

streptococcus pneumoniae-specific crRNA having a sequence shown in SEQ ID NO.9,

haemophilus influenzae specific crRNA with the sequence shown as SEQ ID No.10,

chlamydia pneumoniae specific crRNA having the sequence shown in SEQ ID NO.11,

4. The kit for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids is characterized by comprising an isothermal amplification system, a detection system and a fluorescence detection system of an enzyme-labeled instrument;

the isothermal amplification system comprising the primer combination according to claim 2 as isothermal amplification primer,

the Cas12a detection system comprises the crRNA combination of claim 3,

5. The kit of claim 4, wherein the Cas12a detection system further comprises a Cas12a protein, a ssDNA reporter system comprising ssDNA FQ reporter for fluorescence detection.

6. The kit of claim 5, wherein the ssDNA FQ reporter,5 'end-labeled fluorescent group for fluorescence detection is 6-FAM and the 3' end-labeled quenching group is BHQ1.

7. A method for multiplex detection of streptococcus pneumoniae, haemophilus influenzae, chlamydia pneumoniae and mycoplasma pneumoniae nucleic acids comprising the steps of:

(1) Extracting nucleic acid of a biological sample to be detected;

(2) RPA isothermal amplification and Cas12a detection: amplifying the biological sample nucleic acid obtained in the step (1) in an RPA isothermal amplification system by using the primer combination in the claim 2, carrying out Cas12a detection on the amplified product, and reacting to obtain a Cas12a detection product;

8. The method according to claim 7, wherein in the step (1), the biological sample to be detected is a sputum sample.

9. The method according to claim 7, wherein in the step (2), the amplification conditions are 39℃for 20 minutes; the Cas12a detection conditions were 37 ℃ for 15 minutes.