阅读说明：本技术 一种双标记报告荧光多重病原体核酸检测方法 (Double-label report fluorescent multiple pathogen nucleic acid detection method ) 是由 张誌 李禹� 王月华 于 2019-10-14 设计创作，主要内容包括：本发明涉及一种双标记报告荧光多重病原体核酸检测方法,本发明在现有技术的基础上设计出包括两种荧光基团,以及一种或两种淬灭荧光基团标记的探针,并使用该探针检测核酸,所述被检测核酸选自病原体,本发明所述探针包括两种荧光基团,称为双标记的报告荧光,所述报告荧光选自FAM、HEX/VIC/HEX/、TAMRA、Cy3、Cy5、CY5.5、Texax、ROX,本发明所述探针包括淬灭荧光,选自BHQ1、BHQ2与BHQ3。(The invention relates to a double-label report fluorescent multiple pathogen nucleic acid detection method, which designs a probe comprising two fluorescent groups and one or two quenching fluorescent group labels on the basis of the prior art, and uses the probe to detect nucleic acid, wherein the detected nucleic acid is selected from pathogens, the probe comprises two fluorescent groups, namely double-label report fluorescence, the report fluorescence is selected from FAM, HEX/VIC/HEX/, TAMRA, Cy3, Cy5, CY5.5, Texax and ROX, and the probe comprises quenching fluorescence and is selected from BHQ1, BHQ2 and BHQ 3.)

1. A double-label report fluorescent multiple pathogen nucleic acid detection method is characterized by comprising two fluorescent groups and one or two probes for quenching fluorescent group labels, and using the probes to detect nucleic acid.

2. The detection method according to claim 1, wherein the pathogen is selected from the group consisting of: respiratory pathogens, fever with eruption pathogens, infectious diarrhea, food-borne important pathogens, entomopathogenic diseases, zoonotic pathogens, hepatitis viruses, and virulent infectious disease pathogens.

3. The detection method according to claim 2, wherein the respiratory pathogen is selected from the group consisting of: influenza A virus, influenza B virus, respiratory syncytial virus, human parainfluenza virus, adenovirus, metapneumovirus, bocavirus, coronavirus, rhinovirus, enterovirus, mycoplasma pneumoniae, chlamydia pneumoniae, streptococcus pneumoniae, epidemic meningitis diplococcus, legionella, pertussis, parapertussis, mycobacterium tuberculosis and other viral bacteria and subtypes thereof; the fever with eruption pathogens are selected from: enterovirus, rubella virus, measles virus, mumps virus, herpes virus, typhoid and paratyphoid fever, parvovirus, cytomegalovirus, rickettsia and other virus bacteria and subtypes; infectious diarrhea and food-borne pathogens are selected from: norovirus, sapovirus, rotavirus, astrovirus, vibrio cholerae, diarrheagenic escherichia coli, vibrio parahaemolyticus, campylobacter, shigella, salmonella, staphylococcus aureus, bacillus, pseudomonas and other virus bacteria and subtypes thereof; the insect-borne infectious disease is selected from: bunyavirus, dengue fever virus, yellow fever virus, west nile virus, Zika virus, chikungunya virus, plasmodium and other virus bacteria and subtypes thereof; the zoonosis pathogen and the hepatitis virus are selected from: hepatitis a virus, hepatitis b virus, hepatitis c virus, hepatitis e virus, human immunodeficiency virus; the infectious animal pathogen is selected from: foot and mouth disease, swine plague, pseudorabies, encephalitis B, porcine parvovirus, brucellosis, streptococcosis, eperythrozoonosis, swine plague, swine paratyphoid fever, swine colibacillosis, rabies, porcine reproductive and respiratory syndrome, swine influenza, swine dysentery, swine erysipelas, swine enzootic pneumonia, and swine pox.

4. The detection method according to claim 2, wherein the pathogen is selected from the group consisting of: influenza a virus (FLUA), influenza virus subtype H1N1, influenza virus subtype H3N2, influenza b virus (FLUB), influenza virus subtype BV, influenza virus subtype BY.

5. The detection method according to claim 2, wherein the probe comprises two fluorescent groups, namely a double-labeled reporter fluorescence selected from FAM, HEX/VIC/HEX/, TAMRA, Cy3, Cy5, CY5.5, Texax, ROX, and a quenched fluorescence selected from BHQ1, BHQ2 and BHQ 3; each reporter fluorescence can be a combination of any two labels, FAM, HEX/VIC/Joe, Cy3, Cy5, CY5.5, Texax, ROX.

6. The detection method according to claim 2, said method comprising the steps of:

step 1, sample extraction

The pathogenic sample types comprise swab type, virus culture, bacteria, fungi, tissue, plasma, serum, whole blood, dry blood spot, feces and environmental sample, and the extraction reagent can be used for extracting nucleic acid by a commercial kit, wherein the method can be a silica gel membrane method or a magnetic bead method;

step 2, PCR;

step 3, fluorescence detection;

and 4, performing qualitative calculation.

7. The detection method according to claim 2, wherein the method of step 1 comprises the following steps:

1) adding 30 μ l of clinical swab sample into 50 μ l of nucleic acid extractive solution, mixing, and cracking at 90 deg.C for 4 min;

2) drying blood spot sample, beating 6mm spot, adding 1ml erythrocyte-removed lysate, standing at room temperature for 3min, centrifuging at 8000rpm for 3min, adding 50 μ l nucleic acid extract, mixing, and lysing at 90 deg.C for 4 min;

3) whole blood samples, 1-5 x 10 suggested⁶Adding 50 μ l of nucleic acid extract into leukocyte, mixing, and lysing at 90 deg.C for 4 min;

4) the urine sample is 100 μ l urine sample, centrifuging at 8000rpm, removing supernatant, adding 50 μ l nucleic acid extract into the precipitate, mixing, and cracking at 90 deg.C for 4 min;

5) the feces sample is recommended to be collected into 200 mu l of water sample, the formed feces with the size of soybean is collected, 900 mu l of PBS and 100 mu l of chloroform are added, vortex oscillation is carried out for 5-10 minutes, centrifugation is carried out for 2min at 12000rpm, and supernatant is transferred into another clean and nuclease-free EP tube; adding 30 μ l of supernatant into 50 μ l of nucleic acid extractive solution, and cracking at 90 deg.C for 4 min;

6) centrifuging at 5000rpm for 1-2min, transferring supernatant 30-50 μ l nucleic acid extractive solution, mixing, and cracking at 90 deg.C for 4 min;

7) mixing bacterial fungus culture solution 10 μ l or toothpick with bacterial plaque to 50 μ l nucleic acid extractive solution, and splitting at 90 deg.C for 4 min;

8) taking out the sample adding tube, and performing instantaneous centrifugation to obtain supernatant which is the extracted nucleic acid;

the PCR method of the step 2 comprises the following steps:

1) designing primers and probes according to the conserved and specific regions of the pathogens, wherein the probes can be two report group markers with the same sequence or two standard report group markers with different sequences; and synthesizing with professional companies;

2) according to a report form of a synthetic company, the primer and the probe are diluted to 10-100 mM;

3) and (3) carrying out PCR reaction preparation: PCR buffer, enzyme, Mg2 were prepared⁺The amount of dNTPs and primer probes;

final concentration of PCR buffer: 1 is prepared from

Final concentration of Taq polymerase is 0.1-1U/. mu.l

Final concentration of RT reverse transcriptase: 1-10U/. mu.l

Mg2⁺Final concentration: 1-5mM

dNTPs final concentration: 0.1-0.8mM

Final concentration of primers and probes: 50-500nM

4) The PCR population may be 20. mu.l to 100. mu.l

Wherein, in the step 3, the fluorescence detection method comprises the following steps:

amplification conditions for RNA pathogens the following procedure can be used

Procedure 1

Procedure 2

The procedure for amplification of DNA pathogens is as follows:

wherein, step 4, the qualitative calculation method comprises the following steps:

the qualitative judgment value is as follows

Positive judgment value of DNA pathogen

CT value Result judgment No CT value or CT-0 Negative of 0<CT<≤37.0 Positive for 37.0＜Ct＜40.0 And (4) an ash area.

8. A kit comprising the probe of any one of claims 1 to 7 and reagents related thereto.

9. The kit according to claim 8, further comprising the following components:

1) reaction solution

2) Enzyme mixture

3) Positive control

4) Negative control

Wherein, the reaction solution: including PCR buffer solution final concentration, dNTPs, Mg²⁺Primer and probe for detecting pathogen and nuclease-free water;

wherein, the enzyme mixed solution: comprises Taq polymerase or/and RT reverse transcriptase and enzyme diluent, which are mixed evenly according to a certain proportion;

wherein, the positive control: the artificial pseudovirus with or without nuclease water dilution is subpackaged into 200 ul/count;

wherein, the negative control: nuclease water-free, quality testing and packaging.

10. The method of using the kit of claim 9, comprising the steps of:

detection of DNA pathogens

1) Preparing a reaction system:

taking out the kit, melting at room temperature, performing vortex oscillation for 60 seconds (the enzyme does not need oscillation), and centrifuging at 6000rpm for 10 seconds; calculating the number n of persons needing to prepare a reaction reagent (n is the number of samples +2 tubes of control); the reaction system for each part is prepared as follows:

formulation system

Name of reagent Sample addition (mul)/parts per person ADV reaction solution 19 Taq polymerase 1 Total volume 20

Calculating the usage amount of the reagents, fully and uniformly mixing, centrifuging at 6000rpm for 10 seconds, and subpackaging into n PCR reaction tubes according to the amount of 20 mul;

2) sample adding:

adding 5 mul (to-be-detected sample DNA, negative control and positive control) into a PCR amplification tube respectively, wherein the total reaction amount is 25 mul;

3) and (3) amplification detection:

transferring the PCR amplification tubes after sample adding to a full-automatic fluorescent quantitative PCR detector for amplification detection, wherein the full-automatic fluorescent quantitative PCR detector (RG3000, ABI Prism7500, ABI Prism 7000 and STRATAGENE Mx3000p types) comprises the following amplification programs:

for the ABI Prism7500, ABI Prism 7000 type fully automatic fluorescent quantitative PCR detector, the fluorescent signal is set as: reporter Dye 1: FAM, Quencher Dye 1: NONE, Passive Reference: NONE; the fluorescent PCR amplification procedure was as follows;

4) reaction procedure:

for RNA pathogen detection:

1) preparing a reaction system:

taking out the kit, melting at room temperature, performing vortex oscillation for 60 seconds (the enzyme does not need oscillation), and centrifuging at 6000rpm for 10 seconds; calculating the number n of persons needing to prepare a reaction reagent (n is the number of samples +2 tubes of control); the reaction system for each part is prepared as follows:

preparation of reaction System

Name of reagent Sample addition amount (. mu.)l)/parts per person Reaction solution 18 Enzyme mixture 2 Total volume 20

Calculating the usage amount of the reagents, fully and uniformly mixing, centrifuging at 6000rpm for 10 seconds, and subpackaging into n PCR reaction tubes according to the amount of 20 mul;

2) sample adding:

adding 5 mu L (to-be-detected sample RNA, negative control and positive control) into the PCR amplification tube respectively;

3) and (3) amplification detection:

transferring the PCR amplification tubes after sample adding to a full-automatic fluorescent quantitative PCR detector for amplification detection, performing amplification program of the full-automatic fluorescent quantitative PCR detector (RG3000, ABI Prism7500, ABI Prism 7000 and STRATAGENE Mx3000 p),

procedure 1

Procedure 2

The technical field is as follows:

the invention relates to detection of nucleic acid, in particular to detection of nucleic acid of pathogens such as viruses and bacteria, and further relates to a double-label report fluorescent multiple pathogen nucleic acid detection method.

Background art:

nucleic acid detection, i.e., nucleic acid amplification detection, generally refers to detection techniques that screen for a particular gene by amplifying DNA or RNA, such as Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), transcription dependent amplification reaction (TMA), and the like. The nucleic acid detection reagent is an in vitro diagnostic reagent based on a nucleic acid amplification detection technology, and is currently used for pathogen detection, early diagnosis of specific diseases, type identification of in vivo substances and the like.

The existing multi-pathogen nucleic acid detection technology is mainly a multiplex PCR technology based on the detection of amplified fragment size by agarose gel electrophoresis or the detection of single fluorescence labeling technology based on the fluorescence PCR principle. The multiplex PCR technique based on the size of the amplified fragment detected by agarose gel electrophoresis is time-consuming and easily causes pollution; based on the fluorescent PCR principle, a single fluorescent labeling technology is used for detecting 4 pathogens at most in the same reaction tube, and if more than 5 pathogens are detected, a multi-tube reaction solution needs to be prepared, so that the time, reagents and templates are wasted.

The invention mainly aims to solve the problems of time and labor consumption and easy generation of cross contamination caused by the need of preparing a plurality of reaction solutions or the need of electrophoresis detection for detecting multiple pathogens.

The invention relates to nucleic acid amplification, in particular to a multiplex PCR method for judging a pathogen based on double fluorescent probe labeling and application thereof. The invention is based on the fluorescent PCR technical principle, and develops a series of multi-pathogen nucleic acid detection methods by reporting a fluorescent interpretation result by double markers for each pathogen.

Disclosure of Invention

During PCR amplification, a pair of primers is added, and a specific fluorescent probe is added at the same time, wherein the probe is an oligonucleotide, and two ends of the probe are respectively marked with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous. Commonly used fluorophores are FAM, TET, VIC, HEX. The substance causing fluorescence quenching is called a fluorescence quenching agent. For example, halogen ions, heavy metal ions, oxygen molecules, and nitro compounds, diazo compounds, carboxyl and carbonyl compounds are all common fluorescence extinguishers.

The invention is to design a probe which comprises two fluorescent groups and one or two quenching fluorescent group labels on the basis of the prior art, and detect nucleic acid by using the probe.

The nucleic acid to be detected is selected from pathogens, such as common human and animal infectious diseases, such as respiratory pathogens, fever with eruption pathogens, infectious diarrhea, food-borne pathogens, insect-borne infectious diseases, zoonosis pathogens, hepatitis viruses, virulent infectious disease pathogens, and the like.

The pathogens preferably include respiratory pathogens such as: (influenza A virus, influenza B virus, respiratory syncytial virus, human parainfluenza virus, adenovirus, metapneumovirus, bocavirus, coronavirus, rhinovirus, enterovirus, mycoplasma pneumoniae, chlamydia pneumoniae, streptococcus pneumoniae, epidemic meningitis diplococcus, legionella, pertussis, parapertussis, mycobacterium tuberculosis and other viral bacteria and subtypes thereof). Fever with eruption pathogen (enterovirus, rubella virus, measles virus, mumps virus, herpes virus, typhoid paratyphi, parvovirus, cytomegalovirus, rickettsia and other virus bacteria and subtypes), infectious diarrhea and food-borne important pathogen (norovirus, sheaves such as virus, rotavirus, astrovirus, vibrio cholerae, diarrhea escherichia coli, vibrio parahaemolyticus, campylobacter, shigella, salmonella, staphylococcus aureus, bacillus, pseudomonas and other virus bacteria and subtypes), entomopathogenic infectious disease (bunyavirus, dengue virus, yellow fever virus, west nile virus, Zika virus, chikungunya virus, plasmodium and other virus bacteria and subtypes), human and livestock comorbid pathogen, hepatitis virus (hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, etc.. and subtypes thereof), Human immunodeficiency virus, etc.), animal infectious pathogens (foot and mouth disease, swine fever, pseudorabies, encephalitis B, swine parvovirus, brucellosis, streptococcosis, epierythrozoosis, swine plague, swine paratyphoid, swine colibacillosis, rabies, porcine reproductive and respiratory syndrome, swine influenza, swine dysentery, swine erysipelas, swine enzootic pneumonia, swine pox, etc.).

Preferably, the nucleic acid of the present invention is an influenza virus, preferably an influenza a virus (FLUA), an influenza virus subtype H1N1, an influenza virus subtype H3N2, an influenza virus subtype b (FLUB), an influenza virus subtype BV, an influenza virus subtype BY.

The invention provides a labeled probe, which comprises two fluorescent groups and is called double-labeled reporter fluorescence, wherein the reporter fluorescence is selected from FAM, HEX/VIC/HEX/, TAMRA, Cy3, Cy5, CY5.5, Texax and ROX,

the probe provided by the invention comprises quenching fluorescence and is selected from BHQ1, BHQ2 and BHQ 3.

Each reporter fluorescence of the present invention can be a combination of any two labels, FAM, HEX/VIC/Joe, Cy3, Cy5, CY5.5, Texax, and ROX.

The probe of the present invention is any probe for detecting the nucleic acid of the present invention.

The present invention further provides a method for detecting nucleic acids using the dual-labeled fluorescence reporter probe of the present invention, comprising the steps of:

step 1, sample extraction

Pathogenic sample types include swabs, viral cultures, bacteria, fungi, tissue, plasma, serum, whole blood, dried blood spots, stool, environmental samples, and the like.

The extraction reagent can be used for extracting nucleic acid by a commercialized kit, and the method can be a silica gel membrane or a magnetic bead method, wherein the method adopts a general nucleic acid rapid extraction reagent produced by Beijing Jinhao pharmaceutical Co., Ltd, and comprises the following steps:

1) adding 30 μ l of clinical swab sample into 50 μ l of nucleic acid extractive solution, mixing, and lysing at 90 deg.C for 4 min.

2) Drying blood spot sample, beating 6mm spot, adding 1ml erythrocyte-removed lysate, standing at room temperature for 3min, centrifuging at 8000rpm for 3min, adding 50 μ l nucleic acid extract, mixing, and lysing at 90 deg.C for 4 min.

3) Whole blood samples, 1-5 x 10 suggested⁶Adding 50 μ l of nucleic acid extract into leukocyte, mixing, and lysing at 90 deg.C for 4 min.

4) The urine sample is prepared by centrifuging 100 μ l urine sample at 8000rpm, removing supernatant, adding 50 μ l nucleic acid extractive solution into the precipitate, mixing, and cracking at 90 deg.C for 4 min.

5) The fecal sample is recommended to be collected into 200 mul of water sample, the formed fecal sample is collected into the size of soybean, 900 mul of PBS and 100 mul of chloroform are added, vortex oscillation is carried out for 5-10 minutes, centrifugation is carried out for 2min at 12000rpm, and supernatant is transferred into another clean and nuclease-free EP tube. Mu.l of the supernatant was added to 50. mu.l of the nucleic acid extract and lysed at 90 ℃ for 4 min.

6) Centrifuging at 5000rpm for 1-2min, transferring supernatant 30-50 μ l nucleic acid extractive solution, mixing, and cracking at 90 deg.C for 4 min.

7) Mixing bacterial fungus culture solution 10 μ l or picking a bacterial plaque with toothpick to 50 μ l nucleic acid extractive solution, mixing, and lysing at 90 deg.C for 4 min.

8) The sampling tube is taken out and is subjected to instantaneous centrifugation, and the supernatant is the extracted nucleic acid.

Step 2, PCR

1) The primers and probes are designed according to the conserved and specific regions of the pathogens, and the probes can be two report group markers with the same sequence or two standard report group markers with different sequences.

And synthesized by professional companies.

2) The primers and probes were diluted to 10-100mM according to the company's report.

3) And (3) carrying out PCR reaction preparation: PCR buffer, enzyme, Mg2 were prepared⁺dNTPs and the amount of primer probe.

Final concentration of PCR buffer: 1 is prepared from

Final concentration of Taq polymerase is 0.1-1U/. mu.l

Final concentration of RT reverse transcriptase: 1-10U/. mu.l

Mg2⁺Final concentration: 1-5mM

dNTPs final concentration: 0.1-0.8mM

Final concentration of primers and probes: 50-500nM

4) The PCR population may be 20. mu.l to 100. mu.l

Step 3, fluorescence detection

Amplification conditions

For RNA pathogens the programs in Table 1 or Table 2 may be

TABLE 1 fluorescent PCR amplification procedure 1

TABLE 2 fluorescent PCR amplification procedure 2

The procedure for amplification of DNA pathogens is as follows:

TABLE 3 DNA pathogen amplification procedure

Step 4, qualitative calculation

The qualitative judgment values are shown in tables 4 and 5

TABLE 4 Positive judgment values for RNA pathogen

TABLE 5 Positive judgment values for DNA pathogen

The invention further includes kits comprising the labeled probes of the invention and reagents related thereto.

The kit of the present invention comprises the following components, for example:

1) reaction solution

2) Enzyme mixture

3) Positive control

4) Negative control

In the kit of the invention, the preparation method of the main reagent is as follows:

reaction solution: including PCR buffer solution final concentration, dNTPs, Mg²⁺Primer and probe for detecting pathogen and nuclease-free water. The preparation method comprises the following specific steps:

in a nuclease-free vessel, PCR buffer (containing Mg) was added sequentially²⁺) To a final concentration of 1 ×, dNTPs, primers and probes, and nuclease-free water are added. Mixing for 30-60min, and packaging.

Enzyme mixture liquid: comprises Taq polymerase or/and RT reverse transcriptase and enzyme diluent, which are mixed evenly according to a certain proportion.

Positive control: the artificial pseudovirus with or without nuclease water dilution is packaged into 200 ul/count.

Negative control: nuclease water-free, quality testing and packaging.

The invention further comprises a method for using the kit of the invention:

detection of DNA pathogens

1) Preparing a reaction system:

the kit was removed, thawed at room temperature and vortexed for 60 seconds (enzyme does not need to be vortexed), and centrifuged at 6000rpm for 10 seconds. The number of reaction reagent copies n (n ═ number of samples +2 tube controls) was calculated. The reaction system for each part is prepared as follows:

TABLE 6 formulation system

Name of reagent	Sample addition (mul)/parts per person
		ADV reaction solution	19
Taq polymerase	1
		Total volume	20

Calculating the usage amount of the reagents, fully mixing the reagents, centrifuging at 6000rpm for 10 seconds, and subpackaging the mixture into n PCR reaction tubes according to the amount of 20 mu l.

2) Sample adding:

5 mul (to-be-detected sample DNA, negative control and positive control) are respectively added into a PCR amplification tube, and the total reaction system is 25 mul.

3) And (3) amplification detection:

transferring the PCR amplification tubes after sample adding to a full-automatic fluorescent quantitative PCR detector for amplification detection, wherein the full-automatic fluorescent quantitative PCR detector (RG3000, ABI Prism7500, ABI Prism 7000 and STRATAGENE Mx3000p types) comprises the following amplification programs:

for the ABI Prism7500, ABI Prism 7000 type fully automatic fluorescent quantitative PCR detector, the fluorescent signal is set as: reporter Dye 1: FAM, Quencher Dye 1: NONE, Passive Reference: NONE. The fluorescent PCR amplification procedure is as follows.

4) Reaction procedure:

(fluorescent Signal Collection)

For RNA pathogen detection:

1) preparing a reaction system:

the kit was removed, thawed at room temperature and vortexed for 60 seconds (enzyme does not need to be vortexed), and centrifuged at 6000rpm for 10 seconds. The number of reaction reagent copies n (n ═ number of samples +2 tube controls) was calculated. The reaction system for each part is prepared as follows:

TABLE 7 reaction system preparation

Name of reagent	Sample addition (mul)/parts per person
		Reaction solution	18
Enzyme mixture	2
		Total volume	20

Calculating the usage amount of the reagents, fully mixing the reagents, centrifuging at 6000rpm for 10 seconds, and subpackaging the mixture into n PCR reaction tubes according to the amount of 20 mu l.

2) Sample adding:

and 5 mu L (to-be-detected sample RNA, negative control and positive control) is added into the PCR amplification tube respectively.

3) And (3) amplification detection:

the PCR amplification tubes after sample addition are transferred to a full-automatic fluorescence quantitative PCR detector for amplification detection, and the amplification program of the full-automatic fluorescence quantitative PCR detector (RG3000, ABI Prism7500, ABI Prism 7000 and STRATAGENE Mx3000p types) can be the following programs 1 and 2 in Table 8 and Table 9:

TABLE 8 fluorescent PCR amplification procedure 1

TABLE 9 fluorescent PCR amplification procedure 2

The invention has the following beneficial effects:

1. the time cost of the operator is greatly saved: the prior fluorescence PCR method for detecting multi-pathogen nucleic acid needs to prepare more than one tube of reaction solution, and the invention can detect 6-15 pathogens only by preparing one tube of reaction solution.

2. The cost is saved: the traditional fluorescent PCR method for detecting multi-pathogen nucleic acid needs to prepare more than one tube of reaction solution, so that more raw materials such as enzyme, buffer, dNTPs and the like are needed, and the method can detect 6-15 pathogens only by preparing one tube of reaction solution, so that the reagent cost can be saved.

3. Avoiding sample contamination: the traditional gel electrophoresis method for detecting multiple pathogenic nucleic acids consumes time and is easy to cause laboratory pollution because steps such as uncapping electrophoresis and the like are needed.

4. Double fluorescence judges a pathogen, theoretically avoiding false positive of experimental results.

Detailed Description