TWI482861B - A method and a kit for the identification of pathogens - Google Patents

Description

Pathogen identification method and identification kit

The invention relates to a method for identifying pathogens and an identification kit, and in particular to an identification method and an identification kit for a highly infectious pathogenic agent.

In recent years, the emergence of global infectious diseases and artificial biological diseases has led to the rapid growth of diagnostic techniques. According to the World Health Organization, infectious pathogens are classified according to their ease of transmission, mortality and morbidity, and impact on public health. Among them, Class A pathogens are the most contagious, with high morbidity and high mortality, which have a great impact on public health. Class B pathogens have a slightly lower infectivity and morbidity, but can still be killed without treatment. .

Examples of pathogens of class A are as follows. Bacillus anthracis is susceptible to skin, inhalation and gastrointestinal tract anthrax, with cutaneous anthrax being the most common form and inhalation anthrax having a high mortality rate. Yersinia pestis is the cause of plague and can infect humans and other animals; plague has three forms, namely bubonic plague, sepsis and pneumonic plague, all with high mortality. Rabbit fever bacteria (Francisella tularensis), Burkholderia pseudomallei (Burkholderia mallei), Burkholderia pseudomallei (Burkholderia pseudomallei) and Bayesian Cox bacteria (Coxiella burnetii) can cause zoonotic disease of humans exposed to low infection These doses of these pathogens can cause debilitating or fatal diseases. The initial stage of human infection with these pathogens can cause flu-like symptoms or non-specific manifestations, which can be accompanied by high mortality and rapid spread once the disease occurs.

O157:H7 Escherichia coli O157 (H7), Salmonella species , Shigella species , and Vibrio cholerae are classified as Class B pathogens. It poses a threat to public health through contaminated food, beverages or water. They cause severe gastrointestinal illnesses that are highly contagious at low infectious doses.

Due to the high infectivity, high morbidity and high mortality of the above-mentioned highly infectious pathogens, methods for effective detection and identification are needed to achieve immediate detection and identification of pathogenic species. However, the traditional pathogen identification method requires bacterial culture and biochemical detection. It takes several days to one week, or even one to two months, to perform the next identification. It takes time and is cumbersome and difficult to distinguish. The virulence of the pathogen. In addition, the current rapid real-time genetic identification method can only detect a pathogen, and it is impossible to simultaneously detect multiple pathogens in one sample.

In order to properly treat the disease caused by the pathogen and prevent the outbreak, the most effective strategy at present is to develop a rapid and accurate pathogen diagnosis method.

Accordingly, one aspect of the present invention provides a method for identifying a pathogen comprising the steps of: (a) providing at least one sample of a DNA sequence (DNA sequence); and (b) providing a plurality of specific primers. a pair of specific primer pairs and an internal control oligo, wherein the specific primer pairs respectively comprise a common sequence and a specific sequence, the common sequence is a first random sequence, and comprises a forward common sequence and a reverse common sequence, The specific sequence is the complementary sequence of the gene of the individual pathogen, wherein one of the primers contains a common sequence, a phosphorothioate linkage and a first calibration, and an internal control oligonucleic acid a second random sequence comprising a common sequence; (c) performing a deoxyribonucleic acid amplification reaction, comprising: a first amplification reaction, and a specific primer pair respectively identifying the sequence of the target gene to amplify the target gene to obtain a first An amplification product comprising a target gene sequence and a common sequence, respectively; and a second amplification reaction occurring after the first amplification reaction, the specific primer Identifying a common sequence to amplify the first amplification product to obtain a first amplification product of the first portion and the second portion, wherein the second amplification product of the first portion comprises a phosphorothioate linkage and a first calibration, the second amplification product of the second portion does not comprise a phosphorothioate linkage and a first calibration; (d) the addition of a T7 exonuclease and a deoxyribonucleic acid amplification reaction a product reaction wherein the T7 exonuclease hydrolyzes the second amplification product of the second portion and leaves a second amplification product of the first portion; and (e) detecting the first portion of the first portion of the T7 exonuclease treatment The target gene in the second amplification product Performance.

According to an embodiment of the invention, the step (e) further comprises preparing at least one oligonucleic probe microsphere, wherein each oligo probe microsphere comprises a microsphere body and a probe designed to the target gene. And one of the oligonucleic acid probes designed by the internal control oligonucleic acid sequence; mixing the second amplification product of the first part and the oligonucleic probe microspheres to perform a hybrid reaction, the surface of the oligonucleic probe microspheres The probe recognizes and binds to the target gene in the second amplification product to obtain a hybrid product; adds a second calibration reagent with a fluorescent receptor to the first calibration product of the hybrid product; and detects the microsphere The amount of fluorescence of the fluorescent receptor upon excitation by light to identify the presence of a pathogen in the specimen.

According to another embodiment of the present invention, when the first calibration is biotin, the second calibration with a fluorescent receptor is streptavidin-phycoerythrin (SAPE), and the second Streptavidin in the calibrator, which is used to bind biotin.

According to still another embodiment of the present invention, the specific primer pair of the first amplification reaction has a higher binding temperature to the target gene than the specific primer pair of the second amplification reaction and the common sequence.

According to an embodiment of the invention, wherein the common sequence of the specific primer pair comprises a forward common primer and a reverse common primer, the forward common primer comprises SEQ ID NO: 1, and the reverse common primer comprises SEQ ID NO: 2 . The specific primer pair comprises a forward specific primer and a reverse specific primer, wherein the forward specific primer comprises a forward common sequence, and the reverse specific primer comprises the reverse common sequence, a phosphorothioate linkage and a first calibration Positive specific The primer comprises SEQ ID NO: 3, and the reverse specific primer comprises SEQ ID NO: 4. The internal control oligonucleic acid line comprises SEQ ID NO:5.

According to an embodiment of the invention, the specific primer pair is selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO :28 with SEQ ID NO:29, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:40 And the group consisting of SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 47, and any combination thereof.

According to still another embodiment of the present invention, the second amplification product of the first portion comprises a target gene sequence, a reverse common sequence, a phosphorothioate linkage and the first calibration substance, and the second amplification product of the second portion comprises the target gene Sequence and forward common sequence.

According to another embodiment of the invention, the oligonucleic acid probe comprises SEQ ID NO: 6, and is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO :21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45 a group consisting of SEQ ID NO: 48 and any combination thereof.

Another aspect of the present invention is to provide a method for detecting high infectivity The identification kit of the pathogen comprises at least a specific primer pair, a specific primer pair, an internal control oligo, a T7 exonuclease and at least one oligonucleic probe microsphere. a specific primer pair selected from SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 41. A group consisting of SEQ ID NO: 43 and SEQ ID NO: 44 and SEQ ID NO: 46 and SEQ ID NO: 47, and any combination thereof; a specific primer pair comprising a forward specific primer and a reverse specific a primer, the forward specific primer comprises SEQ ID NO: 3, and the reverse specific primer comprises SEQ ID NO: 4; an internal control oligo nucleic acid comprising SEQ ID NO: 5; an oligonucleic probe probe microsphere comprising SEQ ID NO :6, and selected from SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, Group consisting of SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, and any combination thereof.

The invention combines a multiplex polymerase chain reaction and a suspension microsphere array analysis technology to provide a highly sensitive and highly specific pathogen detection and identification method, which can simultaneously simultaneously target a plurality of bacterial highly infectious pathogenic genes. Detection and identification of pathogenic virulence strains and non-virulence strains, and can be applied to the detection of multiple infections.

(a), (b), (c), (d), (e) ‧ ‧ steps

110‧‧‧DNA sequence

110a‧‧‧First DNA sequence

110b‧‧‧Second DNA sequence

120, 120a, 120b‧‧‧Special primer pairs

120a ₁ , 120b ₁ , 140 ₁ ‧ ‧ common sequence

120a ₁₁ , 120b ₁₁ , 130a , 140 ₁₁ ‧ ‧ forward common sequence

120a ₁₂ , 120b ₁₂ , 130b _1, 140 ₁₂ ‧‧‧ Reverse common sequence

120a ₂ , 120b ₂ ‧‧‧Special sequence

120a ₂₁ , 120b ₂₁ ‧‧‧ Forward specific sequence

120a ₂₂ , 120b ₂₂ ‧‧‧ Reverse specific sequence

130‧‧‧Specific primer pairs

130a‧‧‧ Forward specific primer

130b‧‧‧Reverse specific primer

130b ₁ ‧‧‧Inverse common sequence

130b ₂ ‧‧‧thiophosphate linkage

130b ₃ ‧‧‧First Calibration

140‧‧‧Internal control oligo

150‧‧‧First amplification product

150a‧‧‧ the first target gene amplified fragment

150b‧‧‧Second-labeled gene amplification fragment

160, 160a, 160b, 160c‧‧‧ second amplification product

160a ₁ , 160b ₁ , 160c ₁ ‧‧‧ the second amplification product of the first part

160a ₂ , 160b ₂ , 160c ₂ ‧‧‧ second amplification product of the second part

170a, 170b, 170c‧‧‧ oligonucleic probe microspheres

170a ₁ , 170b ₁ , 170c ₁ ‧ ‧ microsphere body

170a ₂ , 170b ₂ , 170c ₂ ‧‧‧Amino group attached to C12

170a ₃ , 170b ₃ , 170c ₃ ‧‧‧ probe

175‧‧‧Hybrid products

180a‧‧‧Second calibration

180b‧‧‧Fluorescent receptor

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; 2A to 2F are schematic diagrams showing the steps of the pathogen identification method according to another embodiment of the present invention; and FIG. 3 is a diagram showing the fluorescence intensity of each target gene according to an embodiment of the present invention. Fig. 4 is a graph showing the fluorescence intensity of a reference line of each target gene according to an embodiment of the present invention.

Figure 5 is a graph showing the fluorescence intensity of each target gene according to another embodiment of the present invention; and Figures 6A to 6D are diagrams showing the genome set and fluorescence intensity of each target gene according to still another embodiment of the present invention. Diagram of the relationship.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The embodiments disclosed below may be combined or substituted with each other in a beneficial situation, or may be in a Other embodiments are added to the embodiments without further description or description.

Please refer to FIG. 1 , which is a flow chart showing a method for identifying a pathogen according to an embodiment of the present invention. As shown in Fig. 1, the step of detecting a pathogen of the present invention comprises: (a) providing at least one sample of a DNA sequence; (b) providing a plurality of pairs of specific primer pairs, a specific primer pair, and an internal portion; Controlling oligonucleic acid, wherein the specific primer pair comprises a common sequence and a specific sequence, and the common sequence is a first random sequence, and comprises a forward common sequence and a reverse common sequence, and the specific sequence is an individual pathogen a complementary sequence of the target gene, wherein the specific primer pair has a specific primer containing a common sequence, a phosphorothioate linkage and a first calibration, and the internal control oligonucleic acid is a second random sequence containing a common sequence; Performing a deoxyribonucleic acid amplification reaction, comprising: a first amplification reaction, wherein the specific primer pair respectively identifies the sequence of the target gene to amplify the target gene to obtain a first amplification product, which respectively comprises the target gene sequence and a common sequence; and a second amplification reaction, which occurs after the first amplification reaction, and the specific primer pair recognizes the common sequence to amplify the first amplification product, thereby obtaining a second amplification product of a first portion and a second portion, wherein the second amplification product of the first portion comprises a phosphorothioate linkage and a first calibration, and the second amplification product of the second portion does not comprise a phosphorothioate a bond and a first calibration; (d) adding a T7 exonuclease to react with a product of a deoxyribonucleic acid amplification reaction, wherein the T7 exonuclease hydrolyzes the second amplification product of the second portion and leaves a second amplification product of the first portion; and (e) detecting the expression of the target gene in the second amplification product of the first portion obtained after treatment with the T7 exonuclease.

The pathogen referred to herein may be a bacterial highly infectious pathogen, however, the identification of the present invention is not limited thereto. In one embodiment, the bacterial highly infectious pathogen is selected from the group consisting of Bacillus anthracis , Yersinia pestis , Francisella tularensis , Brucella species , Burkholderia mallei , Burkholderia pseudomallei , Coxiella burnetii , O157:H7 Escherichia coli O157:H7, Salmonella Salmonella species ), Shigella species , Vibrio cholerae , and any combination thereof. A plurality of pathogens can exist simultaneously in the same sample, and the detection method provided by the present invention can simultaneously identify a plurality of pathogens from a sample.

Next, in order to further understand the embodiment of the above steps, please refer to FIGS. 2A to 2F, which are schematic diagrams showing the steps of the pathogen identification method according to another embodiment of the present invention.

Step (a) provides DNA of at least one sample, as shown in FIG. 1A, which shows that the DNA sequence 110 of a sample comprises a first DNA sequence 110a and a second DNA sequence 110b, wherein each DNA sequence is A DNA sequence comprising at least one gene that is the target of the pathogen. The method of extracting the DNA of the sample is a conventional technique, and thus is not limited to any method commonly used in laboratories or a commercially available purification kit.

The identification method of the invention can be used for detecting the isolated and purified bacterial colonies and detecting the presence or absence of these pathogens in clinical biological samples, and can also be applied to detecting environmental samples. The bioassay system is selected from the blood or body fluid of human or animal, serum, cerebrospinal fluid, nasal cavity, nasopharyngeal specimen, throat a group consisting of a specimen, a lung flushing fluid, a sputum, an eye specimen, an ear specimen, a genital tract, a pus, a wound, a urine, a rectal swab, a stool, and any combination thereof; and an environmental inspection system Choose from a group of free air, drinking water, soil, food, powder, environmental surfaces, and any combination thereof.

Step (b) is to provide a pair of specific primer pairs, a pair of specific primer pairs, and an internal control oligo, wherein the forward specific primer comprises a forward common sequence, and the reverse specific primer comprises an inverted common sequence, thiophosphate Bond and first calibration. As shown in FIG. 2B, the dedicated primer pair 120 includes a first specific primer pair 120a and a second specific primer pair 120b, wherein each group of dedicated primer pairs 120 has a common sequence 120a ₁ , 120b ₁ and a specific sequence 120a ₂ , 120b _{2 .} The common sequence 120a ₁ , 120b ₁ contains forward common sequences 120a ₁₁ , 120b ₁₁ and reverse common sequences 120a ₁₂ , 120b ₂₁ ; the specific sequences 120a ₂ , 120b ₂ contain forward specific sequences 120a ₂₁ , 120b ₂₁ and reverse specific Sequences 120a ₂₂ , 120b ₂₂ . The 5' end of each positive/inverse primer of a specific pair of primer pairs contains the same positive/negative common sequence, and the common sequence system does not have any random sequence in any known gene, or the common sequence of forward and reverse primers must Not in the same gene. In one embodiment, the size of the common sequence is about 16 bp. The specific sequence is a complementary sequence designed by using the sequence of the target gene as a template, and the target gene of the pathogen is identified in the amplification reaction, thereby amplifying a partial sequence of the target gene. Each pathogen has its own specific target gene, and the identification method of the present invention detects the gene of each pathogen to confirm that the pathogen is present in the sample.

In one embodiment, the specific primer pair is designed according to fourteen of the eleven bacteria, wherein the target gene is selected from the sap gene of Bacillus anthracis , the pag gene and the cap gene, and the plague bacillus ( Yersinia pestis ) predicts the methyltransferase gene and pla gene, the ISFtu2 gene of Francisella tularensis , the alkB gene of Brucella species , Burkholderia Mallei ) IS407A-fliP gene, orf11 gene of Burkholderia pseudomallei , IS1111 gene of Coxiella burnetii , rfb of O157:H7 type Escherichia coli O157:H7 The gene, the trtrC of the Salmonella species , the ttrA gene, the ipaH1.4 gene of the Shigella species , the recA gene of Vibrio cholerae , and any combination thereof. The detection method provided by the invention can simultaneously identify a plurality of pathogens from a sample, so that the desired specific primer pair can be provided according to the pathogenic original to be detected. For example, anthrax (Bacillus anthracis), plague bacillus (Yersinia pestis), rabbit fever bacteria (Francisella tularensis), Burkholderia pseudomallei (Burkholderia mallei), Burkholderia pseudomallei (Burkholderia pseudomallei) and Bayesian test Coxiella burnetii can cause diseases common to humans and animals. Therefore, if the pathogen of common diseases of humans and animals is to be detected, the specific primers designed by the target genes of the above pathogens are simultaneously tested; as for O157:H7 type Escherichia coli O157:H7, Salmonella species , Shigella species , and Vibrio cholerae cause severe gastrointestinal illnesses, so gastrointestinal diseases are detected. The pathogen is detected simultaneously using a specific primer designed by the target gene of the above pathogenic bacteria.

The specific primer pair 130 includes a forward specific primer 130a and a reverse specific primer 130b, and the forward specific primer 130a includes a forward common sequence. The reverse specific primer 130b includes an inverted common sequence 130b ₁ , a phosphorothioate linkage 130b _{2 ,} and a first calibration 130b ₃ . The 5' end of the reverse primer 130b contains four phosphorothioate linkages 130b ₂ which are inserted between the five terminal bases, respectively, and the first end is labeled with the first calibration 130b ₃ . In one embodiment, the first calibration is biotin. In another embodiment, the Tm value of the specific primer pair described above is higher than the Tm value of the particular primer pair. The Tm value is defined as the temperature at which 50% of the DNA molecules change from double strands to single strands when the temperature rises to a certain value, which is called the Tm value. In yet another embodiment, the product of the specific primer pair and the particular primer pair is about 100 to 200 bp in size.

The internal control oligo nucleic acid 140 is a second random sequence comprising a complementary sequence of a forward common sequence and an inverted common sequence, and the amplified product is used as an internal positive control group for subsequent hybridization reactions. In one embodiment, the internal control oligo is about 80 bases in length.

Step (c) performing a deoxyribonucleic acid amplification reaction, as shown in Fig. 2C, wherein the deoxyribonucleic acid amplification reaction is a step of multiplying step (b) to a specific primer pair 120, a specific primer pair 130 and an internal The control oligo nucleic acid 140 and the sample DNA 110 of the step (a) are simultaneously placed in an environment in which the amplification reaction is carried out.

The first amplification reaction is to identify the DNA sequence of the target gene by the specific primer pair 120 to amplify the target gene, and obtain the first amplification product 150, which comprises the first target gene sequence amplified fragment 150a and the second target gene sequence expansion. Increase fragment 150b. The first amplification product 150 has a common end sequence.

After the second amplification reaction occurs in the first amplification reaction, the specific primer pair 130 recognizes the common sequence of the first amplification product to amplify the first amplification product, and obtains the second amplification product 160, which comprises two targets. A second amplification product 160a, 160b of the gene and a second amplification product 160c of the internal control oligo. Since the specific primer pair 130 only reverses the specific primer 130b with the phosphorothioate linkage 130b ₂ and the first calibration 130b ₃ , two second amplification products 160 are available. The second amplification product 160a ₁ , 160b ₁ , 160c _{1 of the} first portion comprises a target gene sequence, a common sequence, a phosphorothioate linkage and a first calibration, and a second amplification product of the second portion 160a ₂ , 160b ₂ 160c ₂ contains only the target gene sequence and the common sequence.

Since the DNA amplification reaction needs to be carried out in two stages, the Tm value of the specific primer pair is designed to be higher than the Tm value of the specific primer pair. In addition, the specific primer pair of the first amplification reaction and the target genes are The bonding temperature is higher than the bonding temperature of the specific primer pair of the second amplification reaction to the common sequence.

The above methods of DNA amplification are well-known techniques, and thus are not limited to polymerase chain reaction (PCR), transcription-mediated amplification assay (TMA), rolling circle amplification (RCA), nucleic acid sequence-based amplification technology (NASBA). And chain displacement amplification (SDA) and the like. Those skilled in the art are aware that when using certain amplification techniques, the primer may require modification, for example, a primer for SDA having a restriction endonuclease recognition site with an additional nucleotide at the 5' end. Similarly, the primers used by NASBA have an RNA polymerase promoter with an additional nucleotide at the 5' end, so modification of the polynucleotide is also encompassed within the scope of the present invention.

Step (d) is the addition of a T7 exonuclease to react with the second amplification product 160. The T7 exonuclease hydrolyzes DNA in the direction from the 5' end to the 3' end, and therefore, the second amplification product of the second portion, 160a ₂ , 160b ₂ , 160c ₂ , is hydrolyzed by the T7 exonuclease. However, the second portion of the second amplification product 160a ₁ , 160b ₁ , 160c _{1 has} a phosphorothioate linkage which prevents hydrolysis of the T7 exonuclease and is therefore left behind, as shown in Figure 2D. Show.

Step (e) detecting the performance of the target gene in the second amplification product of the first portion. In one embodiment, the 2E and 2F diagrams depict the use of suspended microsphere array analysis techniques to detect the expression of the target gene in the amplified product, however, other methods for detecting the expression of the target gene may be used, and It is limited to the above suspension microsphere array analysis method.

The steps of the suspension microsphere array analysis method include the following. First, at least one oligonucleic probe probe microspheres 170a, 170b, 170c are prepared, as shown in Fig. 2E, wherein each set of oligonucleic probe probe microspheres 170a, 170b, 170c comprises a microsphere body 170a ₁ , 170b _{1 , respectively.} , 170c ₁ and a probe 170a ₃ , 170b ₃ , 170c ₃ designed as the complement of the target gene, and the 5' end of the probe carries the amine group 170a ₂ , 170b ₂ , 170c ₂ with C12 as a linker. The probe described above is a complementary sequence designed with the sequence of the gene responsible for the pathogen as a template, and one of the sets is a DNA probe 170c designed with an internal control oligo nucleic acid as a template.

Next, the first portion of the second amplification product 160a ₁ , 160b ₁ , 160c ₁ and the oligonucleic acid probe microsphere 170 are mixed to perform a hybrid reaction, and the probes 170a ₃ , 170b _{3 of the} surface of the oligonuclear probe microsphere, 170c ₃ recognizes and binds to the target gene in the second amplification product to obtain a hybrid product 175. It is worth mentioning that, because the length of the probe is short and the length of the target gene is long, the target gene is easily combined with another gene, so that there is a competition between the probe and the complementary sequence of the target gene. The step of adding T7 exonuclease to hydrolyze the complementary sequence of the target gene can increase the specificity of the hybrid reaction between the probe and the target gene.

Then, with the second calibration object 180b of the fluorescent acceptor 180a, 130b ₃ will be combined with the second calibration object 180a hybrid product of a first calibration object, as shown in FIG. 2F, FIG. In one embodiment, the first calibrator is biotin and the second calibrator is streptavidin, so the second calibrator with a fluorescent acceptor is phycoerythrin-conjugated streptavidin (streptavidin-phycoerythrin, abbreviated as SAPE), in the second calibration, streptavidin is used to bind biotin.

Next, the amount of fluorescence of the fluorescent receptor on the microsphere after excitation by light is detected to identify the pathogenicity of the sample. In one embodiment, the amount of fluorescence after excitation of the fluorescent acceptor on the microsphere is detected by flow cytometry. However, the method of detecting the amount of fluorescence is not limited thereto, and conventional methods for detecting the amount of fluorescence can be applied thereto.

Example

The following examples are presented to illustrate specific aspects of the invention, such that those of ordinary skill in the art to which the invention pertains. These examples are not intended to limit the scope of the invention.

Example 1: Introduction of primer pair and probe

All primer pairs and probes were purchased from Eurofins MWG Operon (Ebersberg, Germany). The specific primer pair has a Tm value of about 48 °C.

For the sequence of the specific primer pair, the specific primer pair, the internal control oligonucleic acid and the internal control probe, please refer to the following list 1. FD stands for the forward primer, RV stands for the reverse primer, and the sequence of the bold plus the bottom line is the common sequence. Wherein the internal control oligonucleic acid sequence comprises a forward common sequence (represented in bold and a bottom line) and a complementary sequence of the reverse common sequence (indicated by the bottom line).

As can be seen from Table 1, the common sequence of the forward specific primer comprises SEQ ID NO: 1, and the common sequence of the reverse specific primer comprises SEQ ID NO: 2. The specific primer pair of the specific primer pair comprises SEQ ID NO: 3; the reverse specific primer comprises SEQ ID NO: 4, and the 5' end thereof contains four phosphorothioate linkages, which are inserted between the 5 terminal bases, respectively. , the end is labeled with biotin. The internal control oligonucleic acid system comprises SEQ ID NO: 5, a complementary sequence of a forward common sequence and a reverse common sequence with a specific primer, and an internal control oligonucleic acid is used as an internal control group. The internal control probe comprises SEQ ID NO: 6, which is the complement of the internal control oligo nucleic acid sequence, and the 5' end of the probe carries an amine group with C12 as a linker.

The specific primer pair is designed according to the fourteen genes of the eleven bacteria. The target gene is the predicted sap gene of Bacillus anthracis , pag gene and cap gene, and predicted methyltransfer of Yersinia pestis . The putative methyltransferase gene and the pla gene, the ISFtu2 gene of Francisella tularensis , the alkB gene of Brucella species , the IS407A-fliP gene of Burkholderia mallei , orf11 gene Burkholderia pseudomallei (Burkholderia pseudomallei), the Bayesian Cox bacteria (Coxiella burnetii) of IS1111 gene, O157: H7 E. coli (Escherichia coli O157: H7) of the rfb gene, Salmonella (Salmonella species) ttrC, the ttrA gene, the ipaH1.4 gene of Shigella species , and the recA gene of Vibrio cholerae .

The specific sequence of the specific primer pair is the complementary sequence of each target gene, please refer to the following list 2, where FD represents the forward primer, RV represents the reverse primer, and the sequence of the bold plus the bottom line is a common sequence.

According to Tables 1 and 2, each pair of specific primer pair positive primers contain a forward common sequence of SEQ ID NO: 1, and the reverse primer contains a reverse common sequence of SEQ ID NO: 2. A specific primer pair designed according to the sap gene sequence of Bacillus anthracis contains SEQ ID NO: 7 and SEQ ID NO: 8, and the probe contains SEQ ID NO: 9; the specific primer pair designed according to the pag gene sequence contains SEQ ID NO: 10 and SEQ ID NO: 11, the probe comprising SEQ ID NO: 12; a specific primer pair designed according to the cap gene sequence comprising SEQ ID NO: 13 and SEQ ID NO: 14, the probe comprising SEQ ID NO :15. Based on Yersinia pestis (Yersinia pestis) of the predicted methyltransferase (putative methyltransferase) gene sequence containing the specific primers SEQ ID NO: 16 and SEQ ID NO: 17, which probe comprising SEQ ID NO: 18; pla gene basis The specific primer pair of the sequence design comprises SEQ ID NO: 19 and SEQ ID NO: 20, the probe of which comprises SEQ ID NO:21. A specific primer pair designed according to the ISFtu2 gene sequence of Francisella tularensis contains SEQ ID NO: 22 and SEQ ID NO: 23, the probe of which contains SEQ ID NO: 24. A specific primer pair designed according to the alkB gene sequence of Brucella species contains SEQ ID NO: 25 and SEQ ID NO: 26, the probe of which contains SEQ ID NO: 27. A specific primer pair designed according to the IS407A-fliP gene sequence of Burkholderia mallei contains SEQ ID NO: 28 and SEQ ID NO: 29, the probe of which contains SEQ ID NO:30. A specific primer pair designed according to the orf11 gene sequence of Burkholderia pseudomallei contains SEQ ID NO: 31 and SEQ ID NO: 32, and the probe contains SEQ ID NO: 33. A specific primer pair designed according to the IS1111 gene sequence of Coxiella burnetii contains SEQ ID NO: 34 and SEQ ID NO: 35, the probe of which contains SEQ ID NO: 36. A specific primer pair designed according to the rfb gene sequence of E. coli O157:H7 ( Escherichia coli O157:H7 ) contains SEQ ID NO:37 and SEQ ID NO:38, the probe of which contains SEQ ID NO:39. A specific primer pair designed according to the trtrC of the Salmonella species , the trrA gene sequence contains SEQ ID NO: 40 and SEQ ID NO: 41, the probe of which contains SEQ ID NO: 42. A specific primer pair designed according to the ipaH1.4 gene sequence of Shigella species comprises SEQ ID NO: 43 SEQ ID NO: 44, the probe of which contains SEQ ID NO: 45. A specific primer pair designed according to the recA gene sequence of Vibrio cholerae contains SEQ ID NO: 46 and SEQ ID NO: 47, the probe of which contains SEQ ID NO:48. Each probe has an amine group with a C12 as a linker at the 5' end.

The reference strains listed in Table 2 are the standard strains of the above 14 pathogenic bacteria as positive standards. The reference strain was purchased from the American Type Culture Collection (ATCC) and first cultured at 37 ° C for 28 to 48 hours in brain-heart infusion agar (BHIA); The Centers for Disease Control (CDC) biosafety guidelines, and the cultivation of such related bacteria must be carried out in the Biosafety Level 3 laboratory.

Example 2: DNA extraction

DNA extraction method According to the QIAamp DNA-mini kit (Qiagen, Valencia, CA) instructions, DNA was precipitated in 100 μl of elution buffer (10 mM Tris-Cl, pH 8.0), and the purified DNA was stored in -20 ° C spare.

Example 3: Multiplex polymerase chain reaction

Multiplex polymerase chain reaction is used to amplify DNA in this implementation In the example, a multiplex polymerase chain reaction is carried out in a volume of 25 μl, which contains 4 μl of DNA template, 60-80 nM of each specific primer pair, 1.0 mM specific primer pair, 50 fM-5 pM of internal control oligo and 1× AmpliTaq Gold PCR master mix (Applied Biosystems, USA). The multiplex polymerase chain reaction step is as follows. First, the enzyme is activated at a temperature of 95 ° C for 15 minutes; then the first amplification reaction is carried out under the conditions of 25 cycles of 94 ° C for 30 seconds, 65 ° C for 1 minute and 72 ° C. second. Next, a second amplification reaction was carried out under the conditions of 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds. The last is 7 minutes at 72 ° C for 1 minute. The product obtained by the amplification reaction is successively subjected to the next step of T7 exonuclease treatment.

Example 4: T7 exonuclease treatment

The T7 exonuclease was treated by adding 20 U of T7 exonuclease (New England Biolabs Inc., Ipswich, MA) with 18 μl of amplification reaction product, water and 1X NEBuffer in a volume of 36 μl. Incubate for 40 minutes under an environment of 25 °C.

Example 5: Suspension microsphere array technology Preparation of oligonucleic acid probe microspheres

In this embodiment, 15 sets of microspheres are required to have different proportions of fluorescent dyes, and each set of microspheres is connected to the 5' end of an oligo-nucleic acid probe, wherein 14 segments of the probe segment oligonucleic acid sequence are pathogenic The complementary sequence of the original amplification product and a probe sequence are complementary sequences of the internal control oligonucleic acid.

The preparation steps are as follows. First, 5 million microspheres are centrifuged at 12000 xg for 4 minutes, and 50 μl of 0.1 M 2-morpholino sulfonic acid (MES; pH 4.5) is used to reconstitute the precipitate. 4 μl of 0.1 mM oligonucleic acid probe (having a modified amino group at the 5' end, a C12-linked amine group and an oligonucleic acid 5 end) was mixed with the above suspension by shaking and ultrasonic shock for about 30 seconds; then, 2.5 was added. L10 mg/ml of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC) to the mixture in the dark at room temperature Incubate for 30 minutes for the coupling reaction, this step needs to be repeated again; then add 1 ml of 0.02% Tween-20 to the mixture and shake for 20 seconds, then centrifuge at 12000 xg for 2 minutes, then 1 ml The precipitate was reconstituted by 0.1% SDS; the pellet was then centrifuged once, reconstituted with 100 μl of Tris-EDTA (pH 8.0) buffer, and the concentration of the microspheres was measured by a hemocytometer. Finally, the coupled microspheres were adjusted to a final concentration of 20,000 microspheres per 1 μl in Tris-EDTA (pH 8.0) buffer and stored at 4 ° C in the dark.

Hybrid reaction

First, 50 μl of a hybrid reaction mixture comprising 3M tetramethyl ammonium chloride, 50 mM Tris-HCl (pH 8.0), 4 mM EDTA (pH 8.0), 0.1% Sarkosyl and Each group of 2,500 oligonucleic acid probe microspheres was mixed with 17 μl of the amplification reaction product, and incubated at 95 ° C for 10 minutes and at 50 ° C for 30 minutes. The mixture was transferred to a 96-well filter disc with 100 μl of hybrid buffer containing 3 M tetramethylammonium chloride, 50 mM Tris-HCl (pH 8.0), 4 mM EDTA (pH 8.0) and 0.1% Sarkosyl) were washed, then 100 μl of hybrid buffer containing 250 ng of SAPE, after 5 minutes of incubation, washed with 100 μl of hybrid buffer and 75 μl of the hybrid buffer was reconstituted.

Example 6: Fluorescence analysis

After the hybrid reaction, analysis was performed by a flow cytometer (Bio-Plex 200 instrument (Bio-Rad) and Bio-Plex Manager (version 5.0)). A red laser (635 nm) was color-coded to identify the microspheres, and a green laser (532 nm) was applied to detect the fluorescent molecules of SAPE on the amplified product. For each set of oligonucleic probe microspheres, the fluorescence value of at least 100 microspheres is included, and the analysis result is expressed by median fluorescence intensity (MFI).

Please refer to FIG. 3, which is a graph showing the fluorescence intensity of each specific target gene relative to the oligonucleic acid probe microspheres after the reaction with the reference line as a positive standard according to an embodiment of the present invention. As shown in Figure 3, the fluorescent signal was weakly detected by the T7 exonuclease-treated product; after the T7 exonuclease treatment, the obtained product had a stronger fluorescent signal. This is because after the T7 exonuclease treatment, the DNA without the cursor is hydrolyzed, so that the DNA becomes a single strand, so that the specific binding of the probe microspheres to the single-stranded DNA can be enhanced, and the specific measurement is enhanced. Fluorescent signal.

In addition, in order to confirm the specificity and accuracy of the method proposed by the present invention, the reference line was used as a positive standard. First, extract the DNA of the reference line as a positive standard, and sequentially perform the amplification of DNA amplification. The T7 exonuclease reaction was carried out, and 15 oligonucleic acid probe microspheres were added to carry out the hybrid reaction. After the reaction, the fluorescence intensity of each target gene relative to the microspheres was detected. For the result, please refer to Fig. 4, A fluorescence intensity diagram showing a reference line of each of the target genes according to an embodiment of the present invention. IC (internal control) represents the internal control group of the oligonucleic probe microspheres; blank control group (no-template control, referred to as NTC) as the negative control group of the sample; Cut-off value is used when establishing this method system, The blank control group (water only) and other samples that did not produce a positive reaction were obtained by taking the fluorescence MFI values obtained by flow cytometry analysis of each group of microspheres from the average value plus 4 standard deviations. The cut-off value, in the present embodiment, is judged to be positive beyond the Cut-off value, and is judged to be negative below the Cut-off value.

According to Fig. 4, it can be seen that the reference strains of Bacillus anthracis ATCC4229 and ATCC14186 can detect the positive sap gene, wherein ATCC4229 contains pXO2, so it is a positive standard for cap gene; ATCC 14186 contains pXO1, so it is pag gene. Positive standard. By analogy, after the DNA of each reference strain is reacted, the specific oligonucleic acid probe microspheres can detect the fluorescent expression (positive result) of the corresponding target gene, while the probes of other non-standard genes are slightly The ball can get a negative result. In addition, each reference line was positive on the microspheres of the internal control group.

Please refer to FIG. 5, which is a graph showing the fluorescence intensity of each target gene according to another embodiment of the present invention. As shown in Figure 5, whether using a set of probe microspheres for testing or multiple sets of probe microspheres for testing, does not affect the analysis of the amount of fluorescence. The results of this experiment show that the identification of the present invention The method can detect at least 11 kinds of pathogens at the same time or detect some of the pathogens, and can be elastically modified according to the situation.

Example 7: Virulence Analysis

In this example, the virulence analysis was directed to Bacillus anthracis and Yersinia pestis. Bacillus anthracis has three standard genes, namely sap gene, pag gene and cap gene. All Bacillus anthracis contains sap gene on its chromosome. However, Bacillus anthracis containing sap gene may not have full virulence and its virulence. Whether the other two plastids have the pag gene and the cap gene. Since the identification method provided by the present invention can simultaneously identify a plurality of disease-causing genes from a sample, it can be judged by the result of the identification whether the pathogen has full virulence. Similarly, Yersinia pestis has two genes for predicting the methyltransferase gene and the pla gene. When two genes are detected in one sample at the same time, it means that the sample is highly virulent. Pathogenic.

Example 8: Quantitative Analysis

Quantitative analysis is to serially dilute the reference line of each consistent pathogen to 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ genome copies, and then according to any embodiment of the present invention The method of identification is tested.

For the quantitative analysis results, please refer to FIGS. 6A to 6D, which are diagrams showing the relationship between the number of sets of genes and the fluorescence intensity of each target gene according to still another embodiment of the present invention. As shown in Figures 6A through 6D, for each pathogen Quantitative analysis, the correlation coefficient is above 0.96. It is worth mentioning that due to the T7 exonuclease treatment, the specificity of the heterozygous reaction is increased, so there is a good quantitative linear result.

Example 9: Sensitivity analysis

The reference lines of each consistent pathogen are serially diluted to be genome copies of ¹ , 5, 10 ¹ , ²⁵ , 50, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ , respectively, and then the present invention The method of identification of any of the examples was tested. When the MFI value of the flow cytometry analysis exceeds the cut-off value of each microsphere group after various sets of genomes, the minimum number of genome sets is used as the minimum detection limit of the relative target gene of the microsphere group (limit of Detection, referred to as LOD).

The results showed that the LOD of the sap gene of Bacillus anthracis was detected as 50 genome sets, the LOD of the Bacillus anthracis cap gene was detected as 25 genome sets, and Bacillus anthracis pag was detected. The LOD of the gene is 25 genome sets, the LOD of the putative methyltransferase gene of the plague bacillus is 25 genome sets, the LOD of the plague pla gene is 25 sets of genomes, and the LOD of the ISFtu2 gene of the rabbit fever pathogen is 100. The number of genome sets, the LOD of the Brucella alkB gene was detected as 50 genome sets, the LOD of the IS407A-fliP gene of B. sinensis was detected as 10 genome sets, and the Orf11 gene of B. sinensis was detected. The LOD is 10 genome sets, the LOD of the ES1111 gene of Cox's disease is detected as 5 genome sets, the LOD of the O157:H7 E. coli rfb gene is detected as 100 genome sets, and the Salmonella ttrC is detected, ttrA LOD gene into the genome copy number 5, LOD ipaH1.4 gene detection of Shigella is 10 genome copy number and LOD recA gene detection of Vibrio cholerae 10 genomes Number.

Identification kit for highly infectious pathogens

Another aspect of the present invention provides an identification kit for a highly infectious pathogen comprising at least one specific primer pair, a specific primer pair, an internal control oligonucleic acid, a T7 exonuclease, and at least one oligonucleic acid probe. Needle microspheres. The specific primer pair is selected from SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 29. SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 41, Groups consisting of SEQ ID NO: 43 and SEQ ID NO: 44 and SEQ ID NO: 46 and SEQ ID NO: 47, and any combination thereof.

A particular primer pair comprises a forward specific primer and a reverse specific primer, the forward specific primer comprises SEQ ID NO: 3, and the reverse specific primer comprises SEQ ID NO: 4. The internal control oligo comprises SEQ ID NO: 5; the oligonucleic probe microsphere comprises SEQ ID NO: 6, and is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18. SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, A group consisting of SEQ ID NO: 45, SEQ ID NO: 48, and any combination thereof.

The identification kit for detecting highly infectious pathogens may further comprise reagents required for extracting the DNA of the sample, reagents required for the amplification reaction, reagents required for the hybridization reaction, and detection of the amount of fluorescence Reagents required or any combination thereof. Other reagents required to identify the kit may be provided as needed, and are not limited to the reagents exemplified above.

The invention provides a highly sensitive and highly specific pathogen detection and identification method, which can detect one or more kinds of bacteria which are simultaneously present in the sample by combining the multiplex polymerase chain reaction and the suspension microsphere array analysis technology. Infectious pathogenic gene, and can distinguish between virulence and non-virulence strains of pathogenic agents, and is applied to the detection of multiple infections. In addition, the identification method of the invention has a rapid reaction (about 6 to 7 hours without the nucleic acid extraction part), saving time, labor, reagent cost and sample dosage; and elastically modifying the content of the rear suspension microsphere array analysis project to avoid The necessary reagents are wasted and measured in 96 or 384-well plate format for high throughput analysis purposes.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.

<110> National Defense Medical College

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(a), (b), (c), (d), (e) ‧ ‧ steps

Claims (19)

A pathogen identification method comprising: (a) providing at least one sample of a DNA sequence (110); (b) providing a complex array of specific primer pairs (120), a specific primer pair (130), and a internal control oligonucleotide (140), wherein each of the plurality of sets (120a) specific primer pair comprises a common sequence (120a ₁₎ and a specific sequence (120a _2), the consensus sequence (120a ₁₎ of the forward common sequence (120a ₁₁₎ is SEQ ID NO: 1 shown in the sequence, the reverse sequence common (120a ₁₂₎ is SEQ ID NO: 2 of the sequence shown, the specific sequence (120a ₂₎ for individual pathogens of target gene a complementary sequence in which the forward specific primer (130a) in the specific primer pair (130) has the forward common sequence shown in SEQ ID NO: 1, and the reverse specific primer (130b) has the SEQ ID NO: 2 a reverse common sequence (130b ₁ ), a phosphorothioate linkage (130b ₂ ), and a first calibration (130b ₃ ), and the internal control oligo (140) comprises a random sequence and the common sequence; Performing a deoxyribonucleic acid amplification reaction, comprising: a first amplification reaction, wherein the specific primer pair (120) respectively recognizes the order of the target genes To amplify the plurality of target gene, to obtain a first amplification product (150) respectively comprising the plurality of target gene sequences and consensus sequences (120a _11, 120a _12); and a second amplification reaction, which is based After the first amplification reaction, the specific primer pair (130) recognizes the common sequence (120a ₁₁ , 120a ₁₂ ) to amplify the first amplification product (150) and the internal control oligo (140) a second amplification product (160) comprising a first portion (160a ₁ , 160b ₁ , 160c ₁ ) and a second portion (160a ₂ , 160b ₂ , 160c ₂ ), wherein the second amplification of the first portion is obtained The product (160a ₁ , 160b ₁ , 160c ₁ ) comprises a phosphorothioate linkage (130b ₂ ) and the first calibration product (130b ₃ ), and the second amplification product of the second portion (160a ₂ , 160b ₂ , 160c) ₂ ) not containing a phosphorothioate linkage and the first calibration; (d) adding a T7 exonuclease to react with the second amplification product (160), wherein the T7 exonuclease hydrolyzes the second moiety a second amplification product (160a ₂ , 160b ₂ , 160c ₂ ), and leaving a second amplification product of the first portion (160a ₁ , 160b ₁ , 160c ₁ ); e) detecting the expression of the target gene in the second amplification product (160a ₁ , 160b ₁ , 160c ₁ ) of the first portion. The method of claim 1, wherein the step (e) further comprises: preparing at least one oligonucleic probe probe microsphere (170a, 170b, 170c), wherein each of the oligonucleic probe microspheres comprises a microsphere body (170a ₁ , 170b ₁ , 170c ₁ ) and a probe (170a ₂ -170a ₃ , 170b ₂ -170b ₃ , 170c ₂ ) containing a complementary sequence of a gene having a C12 as a linker's amino group and an individual pathogen -170c ₃ ), and the oligonucleic probe microspheres have a oligonucleic acid probe (170c _{2 -} 170c ₃ ) containing the internal control oligonucleic acid sequence; and the second amplification product (160a) of the first portion is mixed ₁ , 160b ₁ , 160c ₁ ) and the oligonuclear probe microspheres (170a, 170b, 170c) for performing a hybrid reaction, and the probes on the surface of the oligonucleic probe microspheres recognize and bind the second Amplifying the target gene sequences in the product to obtain a hybrid product; adding a second calibration product (180a) with a fluorescent receptor (180b) and the first calibration product (130b ₃ ) of the hybrid product Combining; and detecting the amount of fluorescence of the fluorescent receptor on the microspheres after excitation by light to identify the pathogens in the samples in. The method of claim 2, wherein the method of detecting the amount of fluorescence is using a flow cytometer. The method of claim 2, further comprising the step of detecting a quantity of pathogens for detecting the amount of fluorescence on the microspheres. The method of claim 2, wherein when the first calibration is biotin, the second calibration with a fluorescent receptor is phycoerythrin-conjugated streptavidin, and the second calibration Streptavidin is used to bind biotin. The method of claim 1, wherein the sample is a bacterial colony, a biological sample or an environmental sample that has been isolated and purified. The method of claim 6, wherein the bioassay system is selected from the group consisting of human or animal blood, body fluids, serum, cerebrospinal fluid, nasal cavity specimens, nasopharyngeal specimens, throat specimens, lung irrigation fluids, sputum, eyes a group consisting of a part of the body, an ear sample, a genital tract, a pus, a wound, a urine, a rectal swab, a stool, and any combination thereof; the environmental inspection system is selected from the group consisting of air, drinking water, soil, food, A group of powders, environmental surfaces, and any combination thereof. The method of claim 1, wherein the first calibration is a living A substance. The method of claim 1, wherein the forward specific primer is SEQ ID NO: 3, and the reverse specific primer is SEQ ID NO: 4. The method of claim 1, wherein the first portion of the second amplification product (160a ₁ , 160b ₁ , 160c ₁ ) comprises the target gene sequence, the reverse common sequence of SEQ ID NO: 2, sulfur a second phosphate amplification product (160a ₂ , 160b ₂ , 160c ₂ ) comprising the target gene sequence and the forward consensus sequence set forth in SEQ ID NO: 1 . The method of claim 1, wherein the sequence of the internal control oligo comprises SEQ ID NO: 5. The method of claim 1, wherein the pathogenic agents are bacterial highly infectious pathogens. The method of claim 12, wherein the bacterial highly infectious pathogens are selected from the group consisting of Bacillus anthracis , Yersinia pestis , Francisella tularensis , Brucella ( Brucella species ), Burkholderia mallei , Burkholderia pseudomallei , Coxiella burnetii , O157:H7 Escherichia coli O157:H7 , a group consisting of Salmonella species , Shigella species , Vibrio cholerae, and any combination thereof. The method of claim 13, wherein the specific primer pairs are designed according to fourteen of the eleven bacteria, wherein the target genes are selected from the sap gene of Bacillus anthracis , pag Gene and cap gene, Yersinia pestis predictive methyltransferase gene and pla gene, ISFtu2 gene of Francisella tularensis , alkB gene of Brucella species , Burkholderia pseudomallei (Burkholderia mallei) of IS407A-fliP gene, Burkholderia pseudomallei (Burkholderia pseudomallei) of orf11 gene, IS1111 gene Bayesian Cox bacteria (Coxiella burnetii) of, O157: H7 E. coli ( Escherichia coli O157:H7 ) rfb gene, Salmonella species ttrC, ttrA gene, Shigella species ipaH1.4 gene, Vibrio cholerae recA gene and any combination thereof The group formed. The method of claim 14, wherein the specific primer pairs are selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 26. SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 47 A group consisting of any combination thereof. The method of claim 13, wherein the oligonucleic probe microspheres comprise SEQ ID NO: 6, and are selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO : 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42 a group consisting of SEQ ID NO: 45, SEQ ID NO: 48, and any combination thereof. The method of claim 1, wherein the specific primer pair of the first amplification reaction has a higher adhesion temperature to the target gene than the specific primer pair of the second amplification reaction and the common sequence temperature. An identification kit for detecting a bacterial highly infectious pathogen comprises: at least one specific primer pair (120), each specific primer pair (120a) comprising a common sequence (120a ₁ ) and a specific sequence (120a ₂ ), The forward common sequence (120a ₁₁ ) in the common sequence (120a ₁ ) is the sequence shown in SEQ ID NO: 1, and the reverse common sequence (120a ₁₂ ) is the sequence shown in SEQ ID NO: 2, the at least one The specific primer pair (120) is selected from SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 16. And SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO : 41, a group consisting of SEQ ID NO: 43 and SEQ ID NO: 44 and SEQ ID NO: 46 and SEQ ID NO: 47, and any combination thereof; a specific primer pair (130) comprising a forward specific primer ( 130a) and a reverse specific primer (130b), the forward special The primer is SEQ ID NO: 3, and the reverse specific primer is SEQ ID NO: 4; an internal control oligo (140) which is SEQ ID NO: 5; T7 exonuclease; and at least one oligo Probe microspheres (170a, 170b, 170c) comprising SEQ ID NO: 6, and selected from SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21. SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO: 48 and any combination thereof, wherein the specific primer pair corresponds to the same bacterial high infectious pathogen target gene as the oligonucleic probe microspheres, respectively. The kit according to claim 18, further comprising: a reagent required for extracting the DNA of the sample, a reagent required for performing the amplification reaction, a reagent required for performing the hybrid reaction, and a reagent for detecting the amount of fluorescence required. Reagents.