CN110079623B

CN110079623B - Method for detecting toxicity of acinetobacter baumannii

Info

Publication number: CN110079623B
Application number: CN201910445920.7A
Authority: CN
Inventors: 黄维; 张敏; 郑超; 邹全明; 章金勇
Original assignee: Shenzhen Peoples Hospital
Current assignee: Shenzhen Peoples Hospital
Priority date: 2019-05-27
Filing date: 2019-05-27
Publication date: 2022-11-11
Anticipated expiration: 2039-05-27
Also published as: CN110079623A

Abstract

The invention relates to a detection method suitable for toxicity of acinetobacter baumannii, and belongs to the technical field of biological detection. Taking a section of specific sequence in the acinetobacter baumannii as a marker, wherein the specific sequence exists in a section of gene sequence comprising 19 open reading frames in the acinetobacter baumannii, and the section of gene sequence comprising the 19 open reading frames is specific to the acinetobacter baumannii with high toxicity; and then, a primer is designed by using a marker specific sequence, and finally, the qualitative detection of high toxicity or low toxicity of the acinetobacter baumannii is completed based on polymerase chain reaction, so that the problems that the acinetobacter baumannii detection method in the prior art can only identify the existence of the acinetobacter baumannii, can not accurately and qualitatively distinguish different toxicity of each strain and the like are solved, and the application values of the marker and the detection method for the toxicity of the acinetobacter baumannii are ensured.

Description

Detection method suitable for toxicity of acinetobacter baumannii

Technical Field

The invention relates to a method for detecting the toxicity of acinetobacter baumannii, in particular to a qualitative detection method for detecting the toxicity of the acinetobacter baumannii into high toxicity or low toxicity, belonging to the technical field of biological detection.

Background

Acinetobacter baumannii is a non-fermented gram-negative bacillus, widely exists in the nature and belongs to conditional pathogenic bacteria. The strain is an important pathogenic bacterium of hospital infection, mainly causes respiratory tract infection, and can also cause bacteremia, urinary system infection, secondary meningitis, operation site infection, ventilator-associated pneumonia and the like. Due to the increasing trend of the drug resistance rate of acinetobacter baumannii to common antibiotics (especially carbapenem antibiotics) year by year, the acinetobacter baumannii attracts the close attention of clinicians and microbiologists. Patients infected by acinetobacter baumannii are mostly old, critically ill, weak in body resistance and patients using broad-spectrum antibiotics for a long time.

The difference in virulence exists among various strains of the acinetobacter baumannii, the infection of the strains with high virulence often leads to high mortality of patients, and the existence of the acinetobacter baumannii can be clinically identified only at present through a biochemical reaction or a mass spectrometry technology, but the difference in virulence among the strains cannot be effectively distinguished, so that the reference value for clinicians is very limited.

The patent document with publication number CN105803041A and name of "a kit for staining capsules of acinetobacter baumannii and a detection method" is disclosed by the national intellectual property office in 2016, month 07 and 27, wherein the patent document discloses: the detection method comprises the following steps: the Acinetobacter baumannii, the Congo red dye solution and the adhesive are mixed and then pushed into a sheet, and the Acinetobacter baumannii capsule is dyed by a Congo red dyeing method. The patent document provides an acinetobacter baumannii capsule staining kit and a detection method, which can be used for quickly evaluating the toxicity of acinetobacter baumannii, are based on a staining method and belong to morphological detection.

Disclosure of Invention

The inventor finds in research that the genome of Acinetobacter baumannii with high toxicity contains a gene sequence comprising 19 open reading frames, the gene sequence contains a unique specific sequence, and a primer is designed by taking the specific sequence as a target (namely a target polynucleotide), so that based on the research result, the specific sequence is taken as a biomarker and is applied to a method for detecting the toxicity of the Acinetobacter baumannii.

The invention aims to overcome the defects of the prior art and provides a detection method suitable for the toxicity of acinetobacter baumannii. In the technical scheme, the specific sequence is used as a marker, a primer is designed by using the marker, and then qualitative detection of high toxicity and low toxicity of acinetobacter baumannii is completed based on polymerase chain reaction, so that the problems that only the existence of the acinetobacter baumannii can be identified, different toxicity of each strain cannot be distinguished accurately and qualitatively and the like in the acinetobacter baumannii detection method in the prior art are solved.

In order to achieve the technical purpose, the following technical scheme is proposed:

the application of a section of specific sequence in the acinetobacter baumannii as a biomarker in a method for detecting the high-toxicity acinetobacter baumannii, wherein the specific sequence exists in a section of gene sequence comprising 19 open reading frames in the acinetobacter baumannii; if the specific sequence is detected to be positive, the toxicity of the acinetobacter baumannii is judged to be high toxicity; otherwise, if the specific sequence is detected to be negative, the toxicity of the acinetobacter baumannii is judged to be low;

the gene sequence comprising 19 open reading frames is shown as SEQ ID NO. 1;

the specific sequence is shown as SEQ ID NO.2.

A detection method suitable for the toxicity of Acinetobacter baumannii comprises the following steps:

s1: taking at least four PCR tubes, respectively numbering the PCR tubes as a positive control tube, a negative control tube, an experimental tube and a blank control tube, and preparing a 20 mu L PCR amplification system; wherein the content of the first and second substances,

adding 15-18 uL PCR reaction solution, 0.5-1.5 uL upstream primer, 0.5-1.5 uL downstream primer and 1-2 uL positive control into the positive control tube;

adding 15-18 uL PCR reaction solution, 0.5-1.5 uL upstream primer, 0.5-1.5 uL downstream primer and 1-2 uL negative control into the negative control tube;

adding 15-18 uL PCR reaction solution, 0.5-1.5 uL upstream primer, 0.5-1.5 uL downstream primer and 1-2 uL acinetobacter baumannii sample to be detected into an experimental tube;

adding 15-18 uL PCR reaction solution, 0.5-1.5 uL upstream primer, 0.5-1.5 uL downstream primer and 1-2 uL sterilized water into a blank control tube;

s2: and (2) respectively centrifuging the PCR amplification system obtained in the step (S1) for 30S at the rotating speed of 3000rpm, and then placing the PCR amplification system in a PCR instrument for amplification, wherein the conditions of amplification reaction are as follows:

reacting for 3min at 94 ℃; then 35 cycles are carried out, wherein the reaction is carried out for 30s at 94 ℃, for 30s at 57-63 ℃ and for 30s at 72 ℃ in sequence; finally, reacting for 5min at 72 ℃, and then keeping at 4 ℃;

s3: and (3) products obtained after the amplification reaction in the step (S2) are subjected to electrophoresis respectively, whether a band appears at the position of 91bp is observed, and result analysis is carried out. If the product after the amplification reaction of the positive control tube has a corresponding strip, and the product after the amplification reaction of the negative control tube and the product after the amplification reaction of the blank control tube have no corresponding strip, the success of the experiment is judged, and then the result of the experiment sample is obtained;

if the product after the amplification reaction of the positive control tube does not have a band correspondingly, or the product after the amplification reaction of the negative control tube has a band correspondingly, or the product after the amplification reaction of the blank control tube has a band correspondingly, the experiment is judged to be unsuccessful, and the experiment needs to be repeated until the experiment is successful, so that the result of the experimental sample is obtained.

Further, in S1, the acinetobacter baumannii sample to be detected is obtained from: inoculating the Acinetobacter baumannii to be detected in a culture medium until the OD600nm of the culture is 0.6-0.8, and taking a culture solution as an Acinetobacter baumannii sample to be detected. Meanwhile, in the step S1, the acinetobacter baumannii sample to be detected in the acinetobacter baumannii culture solution is identified by biochemical reaction or mass spectrum, and the identification technology of the biochemical reaction or the mass spectrum is the existing mature technology; the inoculation, culture, bacteria concentration detection and the like are mature prior art, such as: refer to the laboratory Manual of clinical bacteriology, in rock, et al.

Further, the PCR reaction solution comprises heat-resistant DNA polymerase, tris (hydroxymethyl) aminomethane hydrochloride solution, magnesium chloride solution, potassium chloride solution, triton solution, formamide solution and deoxyribonucleoside triphosphate.

Further, in the 20 μ L PCR amplification system, the concentration of thermostable DNA polymerase is 5U/uL, the concentration of tris hydrochloride solution is 200mmol/L, the concentration of magnesium chloride solution is 20mmol/L, the concentration of potassium chloride solution is 500mmol/L, the volume ratio of triton solution is 0.2%, the volume ratio of formamide solution is 10%, and the concentration of deoxyribonucleoside triphosphate is 10mmol/L.

Further, the upstream primer sequence is as follows: 5 'TTGAGAAGCTAAATTATGGCTCG-3', and the downstream primer sequence is as follows: 5 'GATAGCACAGAATCCATAAAGGAA-3'.

Further, in the 20 muL PCR amplification system, the concentrations of the upstream primer and the downstream primer are both 0.5umol/L; the upstream primer and the downstream primer are used for amplifying a specific sequence SEQ ID NO.2.

Further, the positive control is clinical acinetobacter baumannii strain nucleic acid which contains biomarkers and has high lethality in animal experiments through whole genome sequencing.

Further, the negative control is clinical acinetobacter baumannii strain nucleic acid which does not contain biomarkers and is non-lethal in animal experiments through whole genome sequencing.

Further, the thermostable DNA polymerase is preferably Taq polymerase.

The PCR instrument is a well-established technology in the prior art, for example: dongsheng Longong ETC811.

The electrophoresis is a well-established technique in the prior art, such as: agarose gel electrophoresis.

In the technical scheme, when a PCR amplification system is prepared, the amounts of the PCR reaction solution correspondingly added into the positive control tube, the negative control tube, the experiment tube and the blank control tube are consistent, the amounts of the upstream primer correspondingly added into the positive control tube, the upstream primer correspondingly added into the negative control tube, the amounts of the downstream primer correspondingly added into the experiment tube and the blank control tube are consistent, and the amounts of the acinetobacter baumannii sample to be detected, the negative control tube, the upstream primer correspondingly added into the blank control tube and the sterile water correspondingly added into the blank control tube are consistent.

By adopting the technical scheme, the beneficial technical effects brought are as follows:

1) The invention takes a specific sequence in a gene sequence comprising 19 open reading frames as a marker, and then completes the qualitative detection of high toxicity or low toxicity of the acinetobacter baumannii based on polymerase chain reaction, so as to solve the problems that the acinetobacter baumannii detection method in the prior art can only identify the existence of the acinetobacter baumannii, can not distinguish different toxicity of each strain accurately and qualitatively, and the like;

2) The invention has reasonable design principle, easy result judgment and high accuracy. The inventor applies a comparative genomics method to screen a section of gene segment comprising 19 open reading frames from the genome of Acinetobacter baumannii with high toxicity, and the existence of the gene segment is related to the toxicity of the Acinetobacter baumannii, so a plurality of specific primers are designed based on the specific sequence in the gene segment, and primers with excellent amplification effect for amplifying target polynucleotides are screened out by optimization, namely, the primers are finally limited to an upstream primer and a downstream primer with smaller non-specific reaction, so as to ensure that the reaction of a PCR amplification system is smaller non-specific reaction; finally, the acinetobacter baumannii is qualitatively detected to be high-toxicity or low-toxicity by polymerase chain reaction, and has higher specificity;

3) In the detection method, a PCR amplification system is prepared, then amplification reaction is carried out, and small non-specific reaction is ensured under the conditions of specific temperature, specific time, specific cycle number and the like. Meanwhile, in the preparation of a PCR amplification system, the culture solution (sample) of acinetobacter baumannii to be detected is directly added into an experimental tube, so that the detection process is simple and quick, the qualitative detection of high toxicity or low toxicity of acinetobacter baumannii can be completed only by 1-2 h, the time consumption is short, and the applicability is strong;

4) In the detection method, the PCR reaction solution, the upstream primer, the downstream primer and the Acinetobacter baumannii culture solution are added in sequence, and the substances are added in the sequence due to the large volume difference of the solutions, so that the convenience and operability of the experiment are guaranteed; on the other hand, the solution with smaller volume is not easy to be added into an empty PCR tube, thereby causing false negative, and therefore, the detection accuracy of the acinetobacter baumannii with high toxicity and low toxicity is indirectly improved.

Drawings

FIG. 1 is a flow chart of the detection method of the present invention;

FIG. 2 shows the result of agarose gel electrophoresis of the amplified reaction product after the PCR reaction, wherein lane M: nucleic Acid (DNA) molecular weight standards (Marker); lane 1: the PCR amplification product of the negative control tube; lane 2: PCR amplification products of the positive control tube; lane 3: PCR amplification products of blank control tubes; lanes 4-9: PCR amplification products of the test tubes (samples);

FIG. 3 is a comparison of lethality of positive and negative samples of highly virulent A.baumannii biomarkers in C57BL/6 mice.

Detailed Description

In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, the one clinical isolate corresponds to one case, such as: the 62 clinical isolates corresponded to 62 patients infected with Acinetobacter baumannii. And, the specific number of samples is set at the maximum capacity.

Example 1

s1: taking four PCR tubes, respectively numbering the four PCR tubes as a positive control tube, a negative control tube, an experimental tube and a blank control tube, and preparing a 20 uL PCR amplification system; wherein the content of the first and second substances,

adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL positive control into a positive control tube;

adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL negative control into a negative control tube;

adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL Acinetobacter baumannii sample to be detected into an experimental tube;

adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL sterilized water into a blank control tube;

the sequence of the upstream primer is as follows: 5 'TTGAGAAGCTAAATTATGGCTCG-doped 3',

the downstream primer sequence is: 5 'GATAGCACAGAATCCATAAAGGAA-3';

s2: and (2) respectively centrifuging the PCR amplification system obtained in the step (S1) for 30S under the condition that the rotating speed is 3000rpm, and then placing the system in a PCR instrument for amplification, wherein the conditions of the amplification reaction are as follows:

reacting for 3min at 94 ℃; then 35 cycles are carried out, wherein the reaction comprises the steps of reacting for 30s at 94 ℃, reacting for 30s at 60 ℃ and reacting for 30s at 72 ℃ in sequence; finally, reacting for 5min at 72 ℃, and then keeping at 4 ℃;

s3: and (3) products after the amplification reaction in the step (S2) are subjected to electrophoresis respectively, whether a band appears at the position of 91bp is observed, and result analysis is carried out. If the product after the amplification reaction of the positive control tube has a corresponding strip, and the product after the amplification reaction of the negative control tube and the product after the amplification reaction of the blank control tube have no corresponding strip, the success of the experiment is judged, and then the result of the experiment sample is obtained;

Example 2

On the basis of embodiment 1, the present embodiment is distinguished in that:

when a PCR amplification system is prepared in the step S1, six PCR tubes are taken and respectively numbered as a positive control tube (one), a negative control tube (one), an experimental tube (three) and a blank control tube (one); the PCR amplification system is 20 mu L, wherein,

adding 15uL PCR reaction solution, 1.5uL upstream primer, 1.5uL downstream primer and 2uL positive control into the positive control tube;

adding 15uL PCR reaction solution, 1.5uL upstream primer, 1.5uL downstream primer and 2uL negative control into the negative control tube;

adding 15uL PCR reaction solution, 1.5uL upstream primer, 1.5uL downstream primer and 2uL acinetobacter baumannii sample to be detected into an experimental tube;

adding 15uL of PCR reaction solution, 1.5uL of upstream primer, 1.5uL of downstream primer and 2uL of sterilized water into a blank control tube;

in the step S2, the amplification reaction is carried out for 3min at the temperature of 94 ℃; then 35 cycles, wherein the reaction comprises the steps of reacting for 30s at 94 ℃, reacting for 30s at 57 ℃ and reacting for 30s at 72 ℃ in sequence; finally, the reaction was carried out at 72 ℃ for 5min and then maintained at 4 ℃.

Example 3

On the basis of examples 1-2, the present example differs in that:

when a PCR amplification system is prepared in the step S1, eight PCR tubes are taken and respectively numbered as a positive control tube (one), a negative control tube (one), an experimental tube (five) and a blank control tube (one); the PCR amplification system is 20 mu L, wherein,

adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL positive control into the positive control tube;

adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL negative control into the negative control tube;

adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL Acinetobacter baumannii sample to be detected into an experimental tube;

adding 18uL PCR reaction solution, 0.5uL upstream primer, 0.5uL downstream primer and 1uL sterilized water into a blank control tube;

in the step S2, the amplification reaction is carried out for 3min at the temperature of 94 ℃; then 35 cycles, wherein the reaction comprises the steps of reacting for 30s at 94 ℃, reacting for 30s at 63 ℃ and reacting for 30s at 72 ℃ in sequence; finally, the reaction was carried out at 72 ℃ for 5min and then maintained at 4 ℃.

Example 4

On the basis of examples 1 to 3, the present example differs in that:

during the preparation of the PCR amplification system in the step S1, thirteen PCR tubes are taken and respectively numbered as a positive control tube (two), a negative control tube (two), an experimental tube (seven) and a blank control tube (two); the PCR amplification system is 20 mu L, wherein,

adding 17uL PCR reaction solution, 0.8uL upstream primer, 0.8uL downstream primer and 1.4uL positive control into a positive control tube;

adding 17uL PCR reaction solution, 0.8uL upstream primer, 0.8uL downstream primer and 1.4uL negative control into a negative control tube;

adding 17uL PCR reaction solution, 0.8uL upstream primer, 0.8uL downstream primer and 1.4uL Acinetobacter baumannii sample to be detected into an experimental tube;

adding 17uL PCR reaction solution, 0.8uL upstream primer, 0.8uL downstream primer and 1.4uL sterilized water into a blank control tube;

in the step S2, the amplification reaction is carried out for 3min at the temperature of 94 ℃; then 35 cycles, wherein the reaction comprises the steps of reacting for 30s at 94 ℃, reacting for 30s at 61 ℃ and reacting for 30s at 72 ℃ in sequence; finally, the reaction was carried out at 72 ℃ for 5min and then maintained at 4 ℃.

Example 5

On the basis of examples 1 to 4, further:

in S1, the acinetobacter baumannii sample to be detected is obtained from: inoculating the acinetobacter baumannii to be detected into a culture medium until the OD600nm of the culture is 0.6, and taking the culture solution as an acinetobacter baumannii sample to be detected. Meanwhile, in the step S1, the Acinetobacter baumannii sample to be detected in the Acinetobacter baumannii culture solution is identified by a biochemical reaction or a mass spectrum, and the identification technology of the biochemical reaction or the mass spectrum is the existing mature technology; the inoculation, culture, bacteria concentration detection and the like are mature prior art, such as: refer to the "laboratory Manual of clinical bacteriology, in rock, et al.

Example 6

On the basis of embodiment 5, the present embodiment is distinguished in that:

the OD600nm involved was 0.8.

Example 7

On the basis of examples 5 to 6, the present example differs in that:

the OD600nm involved was 0.7.

Example 8

On the basis of the examples 1-7, further:

in the 20 muL PCR amplification system, the PCR reaction solution comprises heat-resistant DNA polymerase with the concentration of 5U/uL, tris hydrochloride solution with the concentration of 200mmol/L, magnesium chloride solution with the concentration of 20mmol/L, potassium chloride solution with the concentration of 500mmol/L, triton solution with the volume ratio of 0.2%, formamide solution with the volume ratio of 10% and deoxyribonucleoside triphosphate with the concentration of 10mmol/L.

Example 9

On the basis of the embodiment 8, further:

in the 20 mu L PCR amplification system, the concentrations of the upstream primer and the downstream primer are both 0.5umol/L.

Example 10

On the basis of example 9, further:

the positive control is clinical acinetobacter baumannii strain nucleic acid which contains biomarkers and has high lethality rate in animal experiments and is determined by whole genome sequencing; the negative control is clinical acinetobacter baumannii strain nucleic acid which does not contain biomarkers and is non-lethal in animal experiments, and the nucleic acid is determined by whole genome sequencing;

the biomarker is a section of specific sequence in acinetobacter baumannii, the specific sequence exists in a section of gene sequence comprising 19 open reading frames in the acinetobacter baumannii, the gene sequence comprising the 19 open reading frames is shown as SEQ ID No.1, and the specific sequence is shown as SEQ ID No.2.

Example 11

On the basis of the embodiment 10, further:

the thermostable DNA polymerase includes Taq polymerase.

Example 12

The relevance of the biomarker and the high toxicity of acinetobacter baumannii is verified.

1. The whole genome sequences of acinetobacter baumannii with different virulence are compared by using Spine software, a gene sequence (shown as SEQ ID NO. 1) which is located on a chromosome and is specific to the acinetobacter baumannii with high virulence and comprises 19 open reading frames is screened, and a section of specific sequence in the gene sequence is taken as a marker;

2. comparing the whole genome sequence of 62 clinical isolates with a highly virulent acinetobacter baumannii marker sequence by using Parsnp software in a Harvest software package to judge whether a marker exists;

3. principle of design

The mortality rate of the high-toxicity acinetobacter baumannii marker positive sample is obviously higher than that of the negative sample (shown in the following table 1), and the result has statistical significance (chi-square test, p is less than 0.01).

A specific sequence (shown as SEQ ID NO. 2) in a section of high-toxicity acinetobacter baumannii marker gene sequence is screened out by adopting a comparative genomics method, and a specific primer is designed according to the sequence. On the basis, a detection method suitable for the virulence of acinetobacter baumannii is provided (shown in figure 1).

4. Based on polymerase chain reaction, the detection method comprises the following steps:

1) Sampling

Inoculating the acinetobacter baumannii to be detected in a culture medium until the OD600nm of the culture is 0.7, and taking a culture solution as an acinetobacter baumannii sample to be detected;

2) Preparation of PCR amplification System

Taking nine PCR tubes, and numbering as a positive control tube (one), a negative control tube (one), an experimental tube (six) and a blank control tube (one) respectively; preparing a PCR amplification system of 20 muL, wherein,

adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL Acinetobacter baumannii sample to be detected obtained in the step 1) into an experimental tube;

adding 17uL PCR reaction solution, 1uL upstream primer, 1uL downstream primer and 1uL sterile water into a blank control tube;

3) Amplification reaction

Centrifuging each PCR tube subjected to the PCR amplification system prepared in the step 2) for 30s at the rotating speed of 3000rpm, and then placing the tubes in a PCR instrument (Dongsheng Longe ETC 811) for amplification, wherein the conditions of the amplification reaction are shown in the following table 2;

。

the PCR reaction solution comprises Taq polymerase, tris hydrochloride solution, magnesium chloride solution, potassium chloride solution, triton solution, formamide solution and deoxyribonucleoside triphosphate; in a 20 mu L PCR amplification system, the concentration of Taq polymerase is 5U/uL, the concentration of tris hydrochloride solution is 200mmol/L, the concentration of magnesium chloride solution is 20mmol/L, the concentration of potassium chloride solution is 500mmol/L, the volume ratio of triton solution is 0.2%, the volume ratio of formamide solution is 10%, and the concentration of deoxyribonucleoside triphosphate is 10mmol/L.

The sequence of the upstream primer is as follows: 5 'TTGAGAGCTAAATTATGGCTCG-containing 3', and the downstream primer sequence is: 5 'GATAGCACAGAATCCATAAAGGAA-3'; in a 20 mu L PCR amplification system, the concentrations of the upstream primer and the downstream primer are both 0.5umol/L.

5. Judgment and result analysis

After the PCR amplification reaction, each PCR reaction product was subjected to conventional agarose gel electrophoresis, and the band appearing at 91bp was positive (as shown in FIG. 2). In 62 Acinetobacter baumannii clinical isolates which are verified by whole genome sequencing, the sample results of the Acinetobacter baumannii markers with high toxicity are positive; the results of samples without the highly virulent acinetobacter baumannii marker were all negative.

6. Verification of correlation between detection positivity and virulence of high-virulence acinetobacter baumannii marker

And (3) verifying the toxicity of the positive sample of the acinetobacter baumannii marker with high toxicity by using an acinetobacter baumannii pneumonia infection model.

Specifically, 3 Acinetobacter baumannii biomarker positive samples and 4 Acinetobacter baumannii biomarker negative samples are randomly selected according to the proportion of 2.5 multiplied by 10 ⁸ The survival rate of C57BL/6 mice infected with CFU for 6-8 weeks in trachea is observed after 7 days, and the results are shown in FIG. 3, which show that: the survival rate of the mice is 0 and the death rate is 100 percent after 2 days of infection by the positive sample of the biomarker; after 7 days of infection of the biomarker negative sample, the survival rate of the mice is 50 percent, the death rate is 50 percent, and meanwhile, the death rate has significant difference (p) through log-rank test<0.0001). And the obtained high-toxicity acinetobacter baumannii marker positive strain presents high mortality after infecting mice, which indicates that the toxicity of the strain is high.

Furthermore, the clinical isolated strain of acinetobacter baumannii from Anhui proves the application value of the marker, the primer and the detection method for qualitatively detecting the high toxicity of the acinetobacter baumannii. The results show that: the mortality rate of the high-toxicity acinetobacter baumannii marker positive sample is obviously higher than that of the negative sample (as shown in table 3), and the results have statistical significance (chi-square test, p < 0.01).

In a first aspect, the present invention shows a gene sequence (shown in SEQ ID NO. 1) specific to highly virulent A.baumannii having 19 open reading frames;

the second aspect shows that a specific sequence (shown as SEQ ID NO. 2) in the gene sequence (shown as SEQ ID NO. 1) is a marker, and a specific primer is designed based on the specific sequence;

the third aspect shows a detection method suitable for the toxicity of acinetobacter baumannii, under the normal condition, the toxicity of acinetobacter baumannii is weaker, a clinician cannot accurately judge whether a patient uses antibiotics, and the method can quickly and accurately detect whether the acinetobacter baumannii has high toxicity or low toxicity, so that the technical scheme has important reference significance for making a treatment scheme of acinetobacter baumannii infected patients.

The above is only a general embodiment of the present invention, and it should be noted that, for those skilled in the art, several other methods and improvements can be developed without departing from highly virulent acinetobacter baumannii marker sequences, specific primers, etc., which are within the scope of the present invention.

Sequence listing

<110> Shenzhen citizen hospital

<120> detection method applicable to toxicity of acinetobacter baumannii

<130> 2019.5.24

<141> 2019-05-27

<160> 2

<170> SIPOSequenceListing 1.0

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cctgtaatgc cttgtcttat attggtgcgg aaccattatt tattgatgtt tcattgaaga 1560

cactcggttt gtgcccaaag gcattagacg cttatttgca agaaaatgct tttattgatg 1620

accaaggtgt atgtaaacac aaagcaacta ataaacgtat ttctgcaatc gttccaatgc 1680

atacatttgg tcatcctgtt gaaattgatg agcttcaaca ggtcgtaacc aagtggaata 1740

ttgcattagt tgaagacgct gctgaaagtt taggttctta ctacaaaggt aaacataccg 1800

gaacttttgg tctagtttca acacttagct ttaacggaaa taaagtcatc actacaggcg 1860

gcggcggtat ggtcttgtgt caagatgagg aacttggcat aagagcgaag cacattacga 1920

caactgcaaa aataccgcat ccttatgagt tttatcatga tgaaaatggt tttaactacc 1980

gtttaccaaa tttgaatgct gccttaggtt gtgcacaaat ggaaggcttg gaaggtttcc 2040

ttgctaaaaa gcgtgattta gctcagcaat atcaagattt ctttaaagat agtgatattt 2100

catttgttgt ggaacctgaa gactgccgat ctaactattg gttaaatgcc attgtttgca 2160

aagataaagc ccaacgagat ttgattttgg atgaaaccaa ttcaaataag gtgatgactc 2220

gcccaatctg gactttaatg accagattgc caatgtacca aactgcatta caaggcgatt 2280

taactaactc attatggttg gaagaaagag ttgtaaatat tccaagttca gttcctctgg 2340

agtaataaga atgaaaaaaa tagccgtttt tacaggaact cgagctgaat atggtttgct 2400

gtattggtta atgcgagata ttcagcaaga tccagagcta gaattacaaa ttctagctac 2460

ggctatgcat tattcatctg agcacggtga aacttggaaa actattgttc aagatggttt 2520

cgaaattacc gaatcggtag aaatgctact gtcatctgat acatcgtccg ctgttgtgaa 2580

atcaatgggt gtgggcttac ttggatttgc agatgcattg aaacgtatgc aacctgattt 2640

attagttgtt ttgggtgatc gtttcgaagc tttggcagtg acgcaggctg ctttaatcat 2700

gcatattcct gttgcacatt tgcacggtgg tgaaatcact gaaggtgcat atgacgaatc 2760

aattcgtcat gcgatcacca aaatgtctaa tatccatttt gctgcagcag aagaatacaa 2820

aaaacgaatt attcagttag gcgagcaacc agaaaaagta tttaatgtcg gcgcactagg 2880

cttagatcat atccagcgta caacatttaa aaatattgca gaattgagtg aattatatga 2940

ttttgatttt tcaaaaccat attttctcat tacttatcat cctgaaacta atctactaga 3000

agaaaatgtt gcacctttat ttgatgcact taagcaaata aaagatgtca attttgtctt 3060

tagttatcca aatgcagata atggtaatac taacattgtg aaagcaatgc ttgatttaaa 3120

agcacagttg ccagatcgtg tattgcttgt aaagagtttt ggtattcaaa actatttaag 3180

tgttttgaaa aatgctttag caatggtggg taattcttca agtggtctat cagaagctcc 3240

agcattacag gtaccaacag taaatattgg tgatcgtcaa aaaggacgct tacgttgtga 3300

gtctatactt gatgttagat tagacgaaag tgaaatttta gaagcattgc aaaaagcaat 3360

taatttccca gacgatgaat tgagtaaagt tgttccacca ttagggttag gcaatacttc 3420

tcagaaaata attgaactta ttaaaacgac ggattttaaa aagaaggcac cattttatga 3480

tctttaatca agacgaaatt tatgtcattg cagaaattgg tgtaaatcat aatggctctg 3540

tagagttagc aaaagagctt attttaaaag ccaaagaatg cggtgcgaat gctgtcaaat 3600

ttcaaacatt taaagcagat agtttattat ctgatcaaac tgaaatggct gcatatcaaa 3660

aagaaaatac tagttctagt caaagccaat tagagcttgt aaaaagttta gagctcactt 3720

atgagcagac agaagaaata cagaaatttt gtgatgagca tcaaattact tttatttcaa 3780

cgccgtttga ctctgatagt ttgaagttct tggtgaatga gatcgatgta ccttacttga 3840

aagtatcatc tgctgatatt tcaaatttac cgtttctcta tgaaattgca tgttcaaaaa 3900

agcatgtcat tttatctaca ggaacagcta gcctaggtga tattgaacaa gcattatcag 3960

tatttgcttt tgttatcgat aagggaacgg aagttcgacc atctcagcag ttatttagag 4020

aagcttattc taaaatttca gtacgtaaac agttgaaaga acaggtttct atcttacact 4080

gtgtaactca atatccggca ttatttgaag aaagtaatct taaagccatt tcaaccttaa 4140

aaaatgtatt tgatttggca acagggtttt ctgatcatac gcttgatgaa tacgcggcgg 4200

ttattggtgt aagtcttggt gctcgcattt tcgaaaagca tattacatta gataaaacga 4260

tggctggccc agaccatgca gcatcaatgg aaccagatga atttaagcat tatgtcgaaa 4320

ttttgcataa aacatatgca gctttgggcg atggtattaa atttatgcta gagcaagaga 4380

gcgataacta ttacctagta cgtcgtagta ttgtggctaa aacagatatt gcggaagggg 4440

agctgttaac ctctgacaat gtcacaacaa aacgagcagg tcgcgtttgt ttagaaccaa 4500

ataagtattg ggatgttgtt ggtacaaaag caaaacgtag ctttaaaaag aacgatttta 4560

ttgaaattta aatgaaaaaa ctggcgattt ttggttcagg tggctttgca aaagagctag 4620

tagatttagc aatagatcaa ggctaccgcc atatattttt tttagaaaga aatgccaaag 4680

atggtgacac cctacttggc ttccccattt tgccagaatc agcaatatcg acattaacgg 4740

atacggtata tgcgataggt gttgctgatc caaagattcg taaaaaaata tatgaagctt 4800

atcccgattt accttatccc aatttgattc attctcaatc atcacttggc tatggcattc 4860

gagagttgct agaacatagt aaaggtgtgg taattgcggc tggagcgaga attacaaatt 4920

catgtcgttt tggtcatttt attatagtaa gttttaacag cacaattggg cacgactgta 4980

ttctagaaaa ctacgtttct gtcatgccag gtgttaatgt gtctggttgt attcatttga 5040

agcaaggcac ttacattgga acaaatgctg ctattttgcc gggcaaaagt ccagagcaat 5100

tgaaatattt aggtgaaaac tccgttatag gcgcgggtgc tgtggttgtg aaacatacgg 5160

aaccgaataa ggtctatatt ggatcacctg ctaaggaatt aagtcgtgac tagagatgtt 5220

aataatataa ttttgcataa aaatgattca attttgaaag cattagagct acttgattta 5280

tatgctttaa gaattgtttt ggtcgttgat gatcacaatc aattgatcgg aagtattaca 5340

gacggtgata ttcgtagagg gcttttaaaa ggccaagatg tgcatgcagc tgttgaaaca 5400

atcatgcata ctaaccctta tagtattgaa gaaggtagct taaataaccg ccagattttt 5460

gagatcatgc gtgaaaaaag ttatctggca ttaccagtaa ttaaaaataa tcagctagtt 5520

aatatcatta cgcttgatga tttgattcgt aaaaaacgta aagaaaatcc agtttttatt 5580

atggctggag gatttggaac acgtttacgt cctttaacag atacatgccc aaaaccaatg 5640

ttacctgttg gtggtaagcc tttactagaa accattatct caagctttaa aaatcaaggc 5700

ttttataaat tttatatttc tactcactat ttaccagaag tgattaatga gcattttggt 5760

aatggtgaga agtttgacgt acagattcaa tatgtacatg aaactgatcc gcttggaact 5820

ggcggtgcat tgagtttatt gccagcatca gatattaaat tgccattcat tgtgattaat 5880

ggtgatgtac taacaaacat gaattttgaa aaattattgg actttcatga aaagcgcgaa 5940

gctattgcaa cgatgtgcgt gagagaattc caatatcaaa ttccttatgg cgtagttaac 6000

tctgaagata atgtaataca aagcatgact gaaaaaccaa gttatttctt tgatattaat 6060

acaggtattt atgtcatttc ccctgaattg ttggcacaag ttaatgcaca gtttattggt 6120

atgccgacta tcttagaaca gcaaatggaa aagaaaaata aagtcttaag ctatccttta 6180

catgaatatt ggctcgacat cggtcatatg gaagactata accgtgcgca aaaagatatt 6240

catatgttgg gtctagaata atatgaaaaa tattgctgtt attggtttag gtaatattgc 6300

tacccgacat cgtcgtaacc ttaaaatgct ttttcctgaa gcaaaattaa ttgtgatgtc 6360

tgctagtggc cgtattccta aagagccagt gactgatagc gatcatatag cagaaagtgt 6420

tgatgaaatt attcagttgc aagttcagtt tgcaataatt gcctcaccag ctccttttca 6480

tgcccaacat gcactaccac ttattaaagc aggtatccct gtattaatag aaaaaccagt 6540

atcagttacc cagacagatg ctcaagcttt aattgatgca gaagcccagt ataagacacc 6600

tgtagcagta ggttactgtt tgcgctattt accttcagcg cagcaagtac gacaaatgat 6660

acaagagggt gtcattggaa acttatataa tgcatttatt gaaattggtc agtatttacc 6720

agattggcga cctaccaagg attatcgcga aacagtatct gctaaaactg aattgggggg 6780

aggagtccta ttagagctga gtcacgagtt agattatgct caatggattt taggttcgct 6840

tactcctaaa tatgtcgttt tacgtgcttc agaagagctc ggtttagaag tcgaggataa 6900

tgccgatctt ttaatgacaa cagcgaaagg agctgttgta aatatccact tagatttttt 6960

gcagcgtaaa gcacatcgta aatgtcgttt tattggcagt gaaggatgta ttgagtggga 7020

tttaattcga aatgaagttg tgcttattaa ggctaaagag caacaagaaa tttatagcgc 7080

ccctgagtgg gataaaaatc aaatgtattt agaaatggtt accgatttta ttaaaaaaat 7140

aaacggacag ccaaaccaat ctataagtct gcaagaagct gaaagaacag taggcctgat 7200

tgtagagatg aaagaatttg tgacgaaaga atgagactgc ttatatgaga aattttgcat 7260

ttatttttgc tagaggcggc tctaaaggtt tacctggaaa aaatattaaa ccattggcag 7320

gaaaaccgtt attacaatat tcaattgata ccgctttagc atcggatttg attgaacaag 7380

tttttgtttc aacagatgat caggctattg cacaagtcgc tatagaaggt ggagcaattc 7440

ttatagagcg acctgctgaa ctagcaactg atcaaagtcc agaatggttg tcttggcgtc 7500

atgcagttga gtgggcaact gagcactatg gctcatttga tgggtttgtt agtttacctg 7560

caactagtcc attacgtagc caagaggatg tagaggctgc gattttaaaa agacaagcag 7620

agacagctga tatttgtatt gcagttacac ccgcaagtcg cagtccttat ttcaatatgg 7680

ttaagtacaa tgaggctggt tttgttgagc ttgtcaatca gcctgaaggt gaagtatcac 7740

gccgtcaaga tgctcccaaa gtttttgata ttacaacagt agtttatgcg acaacgcctg 7800

agtttgtatt aaatagctat ggtttatttt cagggaaagt tgcgagtatt gaagtaccaa 7860

aagcaagagc tgtagatatt gatgatattt acgattttcg tttagctgaa gctattatta 7920

aaggtgaata attgatgcaa aatttattac agaataaaac ctttttagta gctggtgccg 7980

gtgggttatt aggcacgcgt ttagtggcgg ctatattaga acaaggtgca aaagttattg 8040

cagctgatat aaaccttgaa tctatggcaa ctcgattaag tagtgtagga gttaatacta 8100

caaacgaaaa gttatcgttg gtttccttag atgtaacaaa tgaaacaagt gtaaagttat 8160

ttttttcaac agatcttaaa attgatgggg ctgttaatac aacataccca cggaataaga 8220

cttatggttc gcattttttt gatgtgactc ttgaaagctt taatgagaac ttatcactac 8280

atttaggtag tatgtttttg tttactcagc aatgtgccaa atattttacg caataccaga 8340

ctcctttttc gcttgtaaat atctcatcta tttatggagt agtggctcca aaatttgata 8400

tttataacaa cacgaaaatg accatgcctg ttgagtatgc tgcaattaaa tcagcaacac 8460

tccatttaaa caaatacaca gttgcttatg tcaacgacag ccgattccgc attaattgtg 8520

taagtccagg tggtatcttt gatcatcaac ctgaaccatt tttagaagca tataagagcc 8580

atacccatgg agctggtatg ttagatgtta cagaaatcat aggttcagtt ttattcttac 8640

tttctgaaca atcaagatat gtaacaggtc aaaatattat tgtcgatgat ggcttttcaa 8700

tataacttaa ttatgagtaa tatatatata ggtgattggt aatatgaata ttattaataa 8760

tatagttgta aaattttttt taacaatagt tttttatata atattcctat taactttaga 8820

tatttttcct aataaacacc tttttgctat tttttcattt agtgtttatt tgttaaatag 8880

accaacattt attttatcac ctcggaatat gatatttata tattatttct tgtggtttgg 8940

tttggcacct atatttgcgg atagatataa ttatttagat tattctgata atatagttta 9000

taaatcttat gtttatttgt cttcaagttt tataactatg atattagttt ctatgtgtat 9060

agaaactaaa tacgaaaaaa agttttcttt tataaaagtt gattatagta atgtaagaat 9120

cgatggtaaa aaactaactt ttacttttat atttagtttt cttttttttc ttttgtattt 9180

atttaatact ggaggattta gttactggat agcaaatatt gatcgagctt ttttgactag 9240

gcaaggggca ggagtatact atttaggctt ttcattgttt ttccccgtat ttatattttt 9300

tactagattt aagtataaaa atttaatttt attaagtaca ttaagtttat tagtgatagc 9360

tttaagtcca tttataggta gtaaacaaaa aattatttat tgttttttat tactgttttc 9420

ttccttaatt ttgagaagaa agttgacact aagtaattct ttaatgattg gcttgcccac 9480

aatcttttta tttgtattag ggaattattt tagaaatgtt tcatggatga cattaagcga 9540

tgttgcttca tattcattta attattttga tactttagat tctttattgc tatttttata 9600

taacagacat tacccatttg aaattatgtc agtaggactc cctttaaata aaattgttaa 9660

tttatataca ggtggcgatg aattttttga tgtaagtgca tattatacta atatatactt 9720

tcctgatgct tggaatatac gagcaacagt acaatttcct gtggaagtag atttattttt 9780

aagttttggt ttttgggtgg gattaattcc tttaggaatt tttgttggaa tatattcatt 9840

aattttcatt aaaatgttat ctagtgaaaa aattgtatat ggttttatat ggtttaattt 9900

atttttctat ttgttaagtc acatgcgtgg tggattattt atttggacgg atttatattt 9960

atatccttat cttattttag tatattttct ttttaaagag gttaaagtta atgtgcctaa 10020

aaaataagaa atatgaaatt ataaaaaaag ctattgaaaa tgaaatttca gtagataatt 10080

atctttttga gaagctaaat tatggctcga aaaagctatc tattttcaaa atgctacgat 10140

tcaaagataa aattccttta tggatttctg tgctatcttc tattttaata ccaatttatt 10200

ttgctattga attttttgtt ttttttggaa ggagtagaaa tacatcaaat cttgaaaatg 10260

atcaatattt tttatctttt tctaactctc caaaagtaat tgcaatttat gaagaatatg 10320

cttctgatat agagtttttg caatgtatta ataataaagt ttattgtttt gttggctatt 10380

taagtcctct agttatcctg aagagtttta ttcattcaat tatactttac tttaaaatga 10440

tattttttgt taatattaaa tattatttac acttaacagc aatttttgaa ttaattttat 10500

tttatgaatt tgttattgta ttaaataaaa gaaatataaa taaaatatgt gtggtaaatc 10560

attatgatag gtggttaact gttttgtcaa atattggtaa ttttgatatt gagataatac 10620

agcatggaat tttaacagaa aattatgttg ttaagcataa aataccaaat atttcttttt 10680

taaaagtttt tgataaatct caattgagta tttttctaaa aaatatatta gagggtatac 10740

ctaataaaat tgattatatg aaaattagtt tagatatcaa cttttctgat aaatgtgaag 10800

tattgataat tagtaatcca ttttttataa atcaggaact gttattatat aaattcttga 10860

aagataaagg gattaaagtt ttttttagac ctcatccact ttatattaat gaagaaataa 10920

aaagtgtagt tgaaaatgat gagttatgtt taggaaaaag atttccatat cctaacgttt 10980

gcttatgtaa tgagtctact ttgggtaaag aatatgaagc aatgggtttt aaagtattat 11040

ggtggaatca aaatacagat tttcataaga tatggaattt agttaataat gcgtaatttt 11100

attttatatg gtttagcttc tgcttttaat agaggtggag tttttcttat actcccttta 11160

ctagctgctt tactttcagt tgtagattac ggtaaatttt ctctctatat gacagtagtt 11220

caacttttag tgccgctaat aagtctaaat atttctttat tggtgggtag ggaaatttac 11280

gaaaaaccta ttgtagttaa aagctttgtt gtactattta atagattttc tatttttaca 11340

ataatagttt tttttacttt aaattttttt attagaaata ttattttatt gattttaatt 11400

tatacctttt tggaagctat ttttttggtg aattctacat atattcgctt taagtatggt 11460

gcagatcaat ttttctatgt ttgttttttt aaaattttta ttctttcttt tttattagga 11520

agtttcttta tctatgataa gcagataata gtatcaataa aaaatcttat catgatcttt 11580

tctttttcta atttatcaat tgtatattat cttgtaaaga cctctttttc tttcagtttt 11640

aagagaatgt ttgtaattat aagcaactat aagagtatgc tacttttttc tattgggtta 11700

ttacctcata cactttctca atgggtaagt tcaagttcag accgtttttt tgtcaaactt 11760

tatacaaatg atatagaact agggtactac tcattttcat actctctggc tgctatattt 11820

atgattgtaa atagttcatt agctctcgga atacctcaat tatgtgttaa aaattttatt 11880

tattttaatt ccaaaaaatt ttactcaata ttcttttcac tagtgtcaat cttatgggtt 11940

ctatttttaa ttatttatat tgggatatta tattattttt cagacaagta tgatgttaaa 12000

tatatattta aaattggata tgtaattcta gtagggatgt attttttatc gttctattat 12060

tattattcgt cttttctttt ttatgataga gcatctaaaa agttatctta tattactttt 12120

tttgtggcag ttttaaatgt ttctctagta gtgcttctta ctcctttgtt tggtatacta 12180

ggtactgcct tcagtacatt aatatcctat ttaatatatg ttttaatgtc ctataaacat 12240

gcaagtgtta attatacggt aactaatttg tatgctcctt tatctttcat agtaattatt 12300

tcagtatcta tatttgtttt tttatacttc tttaaaatag gtggtgtgag ttatgtttaa 12360

aaataaggtt ttattaatta ctggtggtac aggatctttt ggaaatgctg tattaaaacg 12420

atttatagat agtgatttta aagaaatccg tatctttagt cgtgatgaaa aaaagcaaga 12480

tgatatgcgc aagaaatatc aatcagctaa gcttaaattt tatatagggg atgttcgaga 12540

ttttaattct gttttaaatg taactagagg tgttgattat atttttcatg ctgcagcact 12600

taagcaggtt ccttcttgcg aattttatcc aatggaagct gttaaaacaa atgtgttggg 12660

gacagaaaat ttattagaag cagccattca aaacaatgtt acaagggtag tttgcttaag 12720

cacagataaa gctgtatatc ctattaatgc aatgggcatt tctaaagcca tgatggaaaa 12780

agttatggtc gcaaagtcac gtaacctaga aggcttagat acagttattt gtggcactcg 12840

ttatggcaat gtgatggctt cccgtggttc tgtgattcca ttgtttgttg atcaaatacg 12900

tattggtaaa tctattacaa taacagatcc gaatatgaca cgttttatga tgactttgga 12960

agatgcggta gacttagttc tctatgcttt cgagcatggt gaaaacgggg atatttttgt 13020

acaaaaagca cctgcggcta ctattgatac tttagctaga gctattactc aattattaga 13080

taaacctgaa catccaattc atattattgg tactcgccat ggagaaaaag cttatgaggc 13140

tttattaagt cgtgaagaaa aagcatgtgc cgaagatctc ggtgattatt tccgtgtacc 13200

agcagaccaa cgcgacttga actacgaaaa gtatgtagaa gatggtgatt taaaaattac 13260

tgagttcgaa gattacaact cacataacac cactcgtcta gatgtagagg gtatgaaaca 13320

attattgctt aaactagatt ttgttcgcgc attaacaaga ggcgaatata tttcaccaga 13380

ggcgtaagca tgaaagtttt agttaccggt tctaatggtt ttattgcaaa aaactttatt 13440

cagtttttat cagagaaggc tgaagttgag attttaaaaa ctcatcgtga aacaacagat 13500

caagagttag agcaatcagt tcttgcttcg gactggattg tgcatttagc gggtgtgaat 13560

cgccctttaa atgaaagtga atttgcggaa ggcaatacca ctttaacaga aaagatcagt 13620

caaattttgc agcaagcaaa taagaaaaca cctgttatct tatcgtcttc tattcaggtc 13680

gagcgtgata acccctatgg taaaagtaag ctaggtggag agcaggcact ggtcactctt 13740

catcaagcag aaggtaatcc ggtttatatt tgccgtttag ccaacgtttt tggtaaatgg 13800

tcacgcccta attataactc tgcagttgca actttttgcc ataatgttgc aaatgatttg 13860

cccttacaaa ttcatgatga aaatgctgtg attcgtcttg tttatgtcga tgatgtagtt 13920

gaaacattct ggaatattct gaatgggcaa caagttgaac aaattttcca agtcgaacct 13980

gaatatcaaa ttaccgtagg tgatttggct aaagttttaa atggctttaa agcatcaaga 14040

gatacactta ttaccgaccg tgtgggaaca ggtcttactc gtgcacttta ttcaacttat 14100

ttgagttttt taaagcctga gcaattcgat tacagtgttc caaaatatgg tgatgctcgc 14160

ggtgtattcg ttgaaatgct gaaaacccca gatgcagggc aattttctta ctttactgct 14220

catccaggaa ttacccgtgg tggtcattat caccatacaa aaactgaaaa gttccttgtt 14280

attaagggta aggcattatt taagtttaag catgttgtaa caggtgaatt ttatgagctt 14340

gaaacacaag gcgatgaacc acgaattgta gaaacggtgc ctggttggac acatgatatt 14400

actaacatcg gtgatgatga gatggtggta atgctatggg caaatgaaat ttttgatcgt 14460

gataagccag atacttatgc gatgccaata acaaattaag agtatagatt tgtgaaaaag 14520

ttaaagttaa tgacagtggt gggcacacgt ccagaaatta tccgtttgtc ttgtgtaatg 14580

gctgcgtgtg atgaatattt tgaccatatt attgttcata ctggtcaaaa ctatgactat 14640

gaattgaatg aaattttctt tactgatttg ggtatccgta aacctgacca tttcttaaat 14700

gcagctggtg cgacaggtgc agaaacgatt ggtaatgtaa ttattgcggt tgataagatt 14760

ttagatgaag tgcaacctga agctttactt gttttagggg acacaaacag ttgtatggca 14820

gtattacctg caaaacgtcg taaaattcca actttccaca tggaagctgg aaaccgttgt 14880

tttgatatgc gagttcctga agaaattaac cgccgaattg tcgatcatac ggctgatatt 14940

aatttaactt acagtacgat tgcccgcgat tatttattgg cagaaggttt accagctgac 15000

ttagtaatca aaacgggtag tccaatgttt gaggtacttc accattacaa agcaaaaata 15060

gaagcttctg atgttcttga gcgtttaaat ttaaaagagc atgaatactt tattgttagt 15120

gctcatcgtg aagaaaacat taactcagat cagaacttct tagatttggt tgaaatgtta 15180

aatgcagttg cagagcgata tcagtaccct gtgattgtat cgacacatcc tcgtacgcgt 15240

aaacgtattg aagagttaaa tattgatttt catcctttga ttcaattatt aaaaccactc 15300

ggttttagtg actacaacaa gttacagctt tcagcaaaag cggcattgtc tgatagtgga 15360

acgattaacg aagaatcatc aatcttgaat ttccctgcac ttaatttgcg tcaagcacat 15420

gagcgtccag aaggaatgga agaagcggct gtaatgatgg taggtttgaa agccgaacgc 15480

attttacaag gtttggcaat tttagaaggt caaactagag gcgaaaatcg tcttttgcgt 15540

ttagttgaag actacagtat gccaaatgtg agtgaaaaag ttgtgcgtat cattatgagt 15600

tacacagatt atgtaaatcg agtgatttgg aagaagtatt gaggttaagg cagtgaagaa 15660

gaaggtctat ttaatcacgg aatattatca tccatatcaa aatacgacag gttacttgtt 15720

aggaaaattg tatgacacct tgaatgtaca aagtgatata gaccttgttc ttattgcaaa 15780

agaggataca aactgtcctc aacatgcaaa tgcacacttt attaaagcta aaaagccaaa 15840

taaggcaagt ttgtttaaaa gatttttata tgaactaatt atcgctttta gttttttaat 15900

gaagactttg caagtagtga aaaaggatag tatcgttttt acaggtacga ctcctatttt 15960

actgcttttt gttttattta ttattaaaaa gtttctaaat tttagatgga tattattagt 16020

ccatgatgta tttcccgaaa atttagcagc tgctaaaatt ttaaaaaaag atcatttttt 16080

atttaaaatc ttaaaaagct tatttgatag gatttatgca agtgctgatg aggtgattgt 16140

cattggcaaa gatatgaaag agttagtaca tcaaaaaacg catcaaagta acaatattac 16200

tgtagtgcag aattggattg actcatctga tattggtaca gagcctaaaa ctaataaccg 16260

aattttacaa gaattgaact ggttggatag tgaaactacg atttttcaat tcttcggtaa 16320

tattggtcgt gtacaaggcg tagaaaatat tctgcatgct attcagaaaa tgaaatatgc 16380

acatttggca aaattcattt ttattggtga tggtgcatat gttgctcgtc ttaaagagca 16440

aatacagtct ttgggatctc aaaatattct ttattatgga tctttagatc agaaagaaaa 16500

aacaactggt ttaaatgcgt gtgatgttgc attgattact ttggctgatg ggatgttagg 16560

tttaggggtt cctagtaaat cttatttttc tatggcggca gataagcctt tgctagcaat 16620

tatggatagg gagtcagaag tagcagatat gattaatact catcatatcg gttgggtagt 16680

cccacctaat aatgaagagg ctttggcaaa acaacttgat gaaattgtgt tagcaaaata 16740

ctgctatgca ttcaattcat caagagaagt attaaatgaa ttttattcag aaaaagtagc 16800

aatgagtaaa attctaaata ttattagaaa atttacttaa gaatttaatt tttaaaatga 16860

atatcttgat tacaggaagt agtggttttg ttggttcata cttatgtgaa tattttacga 16920

caatgccaga atatagtgtt ctggctcaaa ctcgtaaaaa tacacaaaag ttaagtaatg 16980

atataaagac tataactttt gatataaatg atgatctaac ttctttggac ttgagctcat 17040

tagatgtcat tgtgcattgt gctgggcgtg ctcatattat gaaggaagtt gaaagtgatc 17100

cactatctat ttatagaaaa gtaaatgtag aaggaacact gaatttagct aaaaaagcag 17160

ctaaagctgg tgtaaaatga tttatattta tgagtagtat taaagtaaat ggtgaagtga 17220

gtgatcaagt accttttact cctcatgata atattataaa tattaaagat ccttatggat 17280

tatcaaaata tgaagctgaa caatctctat taaatttagc gcaaaactcg atgatggagg 17340

tggtcattat tcgcccagct ttaatctatg ggccaaatgt taaggcaaat tttaaaagta 17400

tgatcaatct ggcggctaaa ggaattcctt tgcctgttgg atgcctcgat aataaaagaa 17460

gtatggtcag tatttataat ttaagcgatt tcattaatat ttgtttatca catcctttag 17520

caaaaaatca ggctttttta attagtgatc aagacgatat aacagttaaa gaattattta 17580

cgaagttagc taaagttcag ggtagaaatc ttattgctgt gcctatacct aaagtattaa 17640

ttaatttttt aactaaactt cttaataaga gtgctgtcgc ttcaagactt tgttctgtat 17700

tgatcgttga cacctcaaag aatatagaac tattaggatg gagagctcct tttagctttg 17760

atgagagctt aaaattgatg ttcaaaaagt aataggtttg atatgaatgt tattatatct 17820

ttcatcattc tttttcttat ttcttttgta ttaactttta gtgttcgaaa atatgcttta 17880

agaaaaaata ttattgattt acctaacttt agaagttcac atatgaagcc cacaccacgt 17940

ggtggtggag tagcaatagt gataagtgtt ctaatggctt tattatatag ctatatatct 18000

cagtttataa gtttgaatga ttttttcatt atagggggga gttgtttgct tattgctctc 18060

cttggttttt gtgatgatca tgctcatatc aattctatgt tgcgcttaag tattcatttt 18120

attactgcaa catttatcgt atttagttta aatggctttg cccaagtctc tgtttttaat 18180

aatttaaact taggtttttt taccaacatt cttgctattc tttttctggt ttggatgctt 18240

aatttatata actttatgga tggtattaat ggtattgcaa gtatcgaagc aatatcagtc 18300

ggactaagtc tagctttatt atacagttgg tatgacatca ataattttat attacttgca 18360

atcacttcta ttgtctgtgg ttttctcgta tggaattttc caaaagctaa aatctttatg 18420

ggagatgtag gtagtagttt tttaggtttt ttattcgcta tattagcttt atatgcttta 18480

aaaatagact ttaagttatt tctagcttgg atcatttgtc taggggtttt tatagtagat 18540

gcaaccttta caattataag gagaatacta agaggggaga aaatatatca agctcatcga 18600

agccacgctt atcaatacgc tgctaggtat tttgatagtc atacacccgt aactttagca 18660

gttttaatca ttaatcttat atggctctta ccttgtgcat tttttgtatt aaataaagca 18720

atttctccat tagtcggttt aataatcgca tattttcctt taataatttt agctatttat 18780

tttaatgcag gacagttaga acataaaact taa 18813

<210> 2

<211> 1086

<212> DNA

<213> Acinetobacter baumannii

<400> 2

atgtgcctaa aaaataagaa atatgaaatt ataaaaaaag ctattgaaaa tgaaatttca 60

gtagataatt atctttttga gaagctaaat tatggctcga aaaagctatc tattttcaaa 120

atgctacgat tcaaagataa aattccttta tggatttctg tgctatcttc tattttaata 180

ccaatttatt ttgctattga attttttgtt ttttttggaa ggagtagaaa tacatcaaat 240

cttgaaaatg atcaatattt tttatctttt tctaactctc caaaagtaat tgcaatttat 300

gaagaatatg cttctgatat agagtttttg caatgtatta ataataaagt ttattgtttt 360

gttggctatt taagtcctct agttatcctg aagagtttta ttcattcaat tatactttac 420

tttaaaatga tattttttgt taatattaaa tattatttac acttaacagc aatttttgaa 480

ttaattttat tttatgaatt tgttattgta ttaaataaaa gaaatataaa taaaatatgt 540

gtggtaaatc attatgatag gtggttaact gttttgtcaa atattggtaa ttttgatatt 600

gagataatac agcatggaat tttaacagaa aattatgttg ttaagcataa aataccaaat 660

atttcttttt taaaagtttt tgataaatct caattgagta tttttctaaa aaatatatta 720

gagggtatac ctaataaaat tgattatatg aaaattagtt tagatatcaa cttttctgat 780

aaatgtgaag tattgataat tagtaatcca ttttttataa atcaggaact gttattatat 840

aaattcttga aagataaagg gattaaagtt ttttttagac ctcatccact ttatattaat 900

gaagaaataa aaagtgtagt tgaaaatgat gagttatgtt taggaaaaag atttccatat 960

cctaacgttt gcttatgtaa tgagtctact ttgggtaaag aatatgaagc aatgggtttt 1020

aaagtattat ggtggaatca aaatacagat tttcataaga tatggaattt agttaataat 1080

gcgtaa 1086

Claims

1. A non-diagnostic detection method suitable for the virulence of Acinetobacter baumannii, which is characterized by comprising the following steps:

s1: taking at least four PCR tubes, numbering the PCR tubes as a positive control tube, a negative control tube, an experiment tube and a blank control tube respectively, and preparing a 20 mu L PCR amplification system; wherein the content of the first and second substances,

adding 15-18 muL of PCR reaction solution, 0.5-1.5 muL of upstream primer, 0.5-1.5 muL of downstream primer and 1-2 muL of positive control into a positive control tube;

adding 15-18 muL PCR reaction solution, 0.5-1.5 muL upstream primer, 0.5-1.5 muL downstream primer and 1-2 muL negative control into a negative control tube;

adding 15-18 mu L of PCR reaction solution, 0.5-1.5 mu L of upstream primer, 0.5-1.5 mu L of downstream primer and 1-2 mu L of acinetobacter baumannii sample to be detected into an experimental tube;

adding 15-18 muL of PCR reaction solution, 0.5-1.5 muL of upstream primer, 0.5-1.5 muL of downstream primer and 1-2 muL of sterilized water into a blank control tube;

the sequence of the upstream primer is as follows: 5 'TTGAGAGCTAAATTATGGCTCG-containing 3',

the sequence of the downstream primer is as follows: 5 'GATAGCACAGAATCCATAAAGGAA-3';

s2: centrifuging the PCR amplification system obtained in the step S1 for 30S respectively under the condition that the rotating speed is 3000rpm, and then placing the system in a PCR instrument for amplification;

s3: respectively carrying out electrophoresis on products subjected to the amplification reaction in the step S2, observing whether a band appears at a position of 91bp correspondingly, and carrying out result analysis;

2. The method for detecting acinetobacter baumannii virulence suitable for non-diagnostic purposes according to claim 1, wherein in S1, the acinetobacter baumannii sample to be detected has an OD600nm of 0.6-0.8.

3. The method for detecting the virulence of acinetobacter baumannii for non-diagnostic purposes according to claim 1, wherein the PCR reaction solution comprises thermostable DNA polymerase, tris hydrochloride solution, magnesium chloride solution, potassium chloride solution, triton solution, formamide solution, and deoxyribonucleoside triphosphates.

4. The non-diagnostic purpose detection method suitable for Acinetobacter baumannii virulence according to claim 3, characterized in that in the PCR amplification system, the concentration of thermostable DNA polymerase is 5U/μ L, the concentration of tris hydrochloride solution is 200mmol/L, the concentration of magnesium chloride solution is 20mmol/L, the concentration of potassium chloride solution is 500mmol/L, the volume ratio of triton solution is 0.2%, the volume ratio of formamide solution is 10%, and the concentration of deoxyribonucleoside triphosphate is 10mmol/L.

5. The non-diagnostic detection method for Acinetobacter baumannii virulence according to claim 1 or 4, wherein the concentration of the upstream primer and the concentration of the downstream primer are both 0.5 μmol/L in the PCR amplification system.

6. The method of claim 3 or 4, wherein said thermostable DNA polymerase comprises Taq polymerase.

7. The method of claim 1, wherein the amplification reaction is carried out under conditions selected from the group consisting of: reacting for 3min at 94 ℃; then 35 cycles are carried out, wherein the reaction is carried out for 30s at 94 ℃, for 30s at 57-63 ℃ and for 30s at 72 ℃ in sequence; finally, the reaction was carried out at 72 ℃ for 5min and then maintained at 4 ℃.