CN117512157A

CN117512157A - Primer probe combination for multiplex digital PCR detection of Proteus, kit and application thereof

Info

Publication number: CN117512157A
Application number: CN202311804256.3A
Authority: CN
Inventors: 侯俊豪; 葛光君; 王军
Original assignee: Zhenzhun Bio Technology Co ltd
Current assignee: Zhenzhun Bio Technology Co ltd
Priority date: 2023-12-26
Filing date: 2023-12-26
Publication date: 2024-02-06

Abstract

The invention provides a primer probe combination for multiplex digital PCR detection of Proteus, which is used for detecting Proteus and simultaneously distinguishing general Proteus and/or Proteus mirabilis and/or Proteus pensonii and/or Proteus hausensis and/or Proteus viscosus, and the nucleotide sequence of the primer is shown as SEQ ID NO:2 and SEQ ID NO: 4. The invention utilizes a digital PCR platform to combine multiple TaqMan probe technology, and realizes a detection method for quantitatively detecting the Proteus, the Proteus vulgaris, the Proteus mirabilis, the Proteus pensonii, the Proteus hausensis and the Proteus viscosus with high efficiency, accuracy, sensitivity and high specificity. The rapid detection of the bacillus proteus in urine, sputum and other samples is realized, the culture is not needed in the detection process, the detection period is short, the manpower is greatly reduced, and the detection efficiency is improved.

Description

Primer probe combination for multiplex digital PCR detection of Proteus, kit and application thereof

Technical Field

The invention belongs to the technical field of pathogen detection, and particularly relates to a primer probe for multiplex digital PCR detection of Proteus, a kit and application thereof.

Background

Proteus is a gram-negative bacterium, which has 5 common species: proteus vulgaris (P.v), proteus mirabilis (Proteus mirabilis, P.m), proteus pennei (P.p), proteus hauseri (P.h) and Proteus viscosus (Proteus myxofaciens, P.my), wherein Proteus mirabilis and Proteus mirabilis are relatively closely related to the clinic. Proteus is a conditional pathogenic bacterium, most of which are secondary infections, and most of them are clinically related to food poisoning, urinary tract infection, respiratory tract infection, wound infection, acute gastroenteritis, septicemia and the like, and general Proteus and Proteus mirabilis are secondary to Escherichia coli in urinary system infection. Therefore, the method for detecting the bacillus proteus rapidly, sensitively, accurately, efficiently and specifically can quantitatively detect the bacillus proteus, and has important significance for food safety and public health safety.

The bacteria culture identification is the most important and traditional method for identifying bacteria, and the colony of the Proteus on nutrient agar and blood agar plates can appear migration and growth phenomena; the colony center appears black on the SS agar selection medium; lactose does not ferment on the intestinal selection medium and shows colorless colonies. Simultaneously, biochemical tests are carried out, and the positive of the proteus to phenylalanine, urease, hydrogen sulfide and the like is taken as an identification basis. Complicated operation, insensitivity and easy occurrence of false negative results; the time consumption is long, and the clinical judgment basis cannot be timely given; the culturing process is easy to cause pollution, and pathogenic bacteria are diffused. In the prior art, serological diagnosis is also adopted, and test tube quantitative suspicious set test is carried out by utilizing antigens specific to the Proteus and antisera of a patient, but the method has insufficient specificity, false positive results are easy to occur, the sensitivity of the reaction can be reduced, and the preparation time of the antisera is longer. In addition, in the prior art, molecular biological diagnosis is also adopted, which relates to conventional PCR, fluorescent quantitative PCR, loop-mediated isothermal amplification (LAMP) and the like, a primer probe is designed aiming at a conserved sequence or a specific gene of the Proteus, and whether the sample contains the Proteus can be rapidly, sensitively and specifically detected after nucleic acid extraction, but the method can not absolutely quantify the number of the Proteus in the sample.

Digital PCR (dPCR) generally comprises two parts of content, PCR amplification and fluorescent signal analysis. In the PCR amplification stage, unlike conventional techniques, digital PCR generally requires dilution of a sample to a single molecule level and an average partitioning into several tens to several tens of thousands of units for reaction. Unlike the method of qPCR for real-time fluorescence measurement of each cycle, the digital PCR technique is to collect the fluorescent signal of each reaction unit after amplification is completed. And finally, calculating to obtain the original concentration or content of the sample through a direct counting or poisson distribution formula.

At present, various methods for diagnosing the proteus are reported at home and abroad, and mainly comprise the following steps: biochemical identification, general PCR and fluorescent quantitative PCR. However, there is no method for rapidly detecting Proteus infection and accurately distinguishing 5 Proteus species in Proteus. In the existing method, the biochemical identification method has the defects of complicated operation, long time consumption and poor specificity; however, the conventional PCR and fluorescent quantitative PCR methods which have been established at present can only distinguish Proteus mirabilis from Proteus, and can not distinguish and quantify Proteus hausensis from Proteus viscosus. As can be seen, the existing Proteus detection techniques have major drawbacks.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a primer probe set and a detection method for detecting the proteus in a sample by using chip type digital PCR, wherein the primer probe set can detect the proteus, the general proteus, the proteus mirabilis, the proteus penmansoni and the proteus viscosus, and can detect the proteus in the sample rapidly, simply, sensitively, specifically and absolutely quantitatively.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the invention provides a primer probe combination for multiplex digital PCR detection of Proteus, which is used for detecting Proteus and simultaneously distinguishing multiplex digital PCR detection of general Proteus and/or Proteus mirabilis and/or Proteus pensonii and/or Proteus hausensis and/or Proteus viscosus, and the nucleotide sequence of the primer is as follows:

SEQ ID NO：2 5’-ATCATGAACGTTCTGGGTACACC-3’

SEQ ID NO：4 5’-GATCGAGCACTCAGGTTACTCTG-3’。

as a preferred embodiment, the nucleotide sequence of the probe comprises: the nucleotide sequence of the probe of the Proteus is shown as SEQ ID NO:5 is shown in the figure; the nucleotide sequence of the probe of the general Proteus is shown as SEQ ID NO:6 is shown in the figure; the nucleotide sequence of the Proteus mirabilis probe is shown in SEQ ID NO: shown in figure 7; the nucleotide sequence of the probe of the Proteus pensonii is shown as SEQ ID NO: shown as 8; the nucleotide sequence of the probe of the Proteus hao is shown as SEQ ID NO: shown as 9; the nucleotide sequence of the probe of the Proteus viscosus is shown as SEQ ID NO: shown at 10.

As a preferred embodiment, the probe labels a fluorescent group and a quenching group, respectively, the fluorescent group being selected from any one of ATTO, FAM, HEX, ROX, CY or CY 5.5; the quenching group is selected from any one of BHQ1, BHQ2, BHQ3 or MGB. The fluorophores selected by the Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii and Proteus viscosus are different.

As a preferred embodiment, when the quenching group is selected from MGB, the fluorescent group is preferably FAM or HEX.

As a preferred embodiment, the probe of the genus proteus is labeled with a fluorescent group ATTO at the 5 'end and a quenching group BHQ1 at the 3' end; the 5 'end of the probe of the general Proteus is marked with a fluorescent group FAM, and the 3' end is marked with a quenching group MGB; the 5 'end of the probe of the Proteus mirabilis is marked with a fluorescent group HEX, and the 3' end is marked with a quenching group MGB; the 5 'end of the probe of the Proteus pensonii is marked with a fluorescent group ROX, and the 3' end is marked with a quenching group BHQ2; the 5 'end of the probe of the Haofacillus is marked with a fluorescent group CY5, and the 3' end is marked with a quenching group BHQ2; the 5 'end of the probe of the Proteus viscosus is marked with a fluorescent group CY5.5, and the 3' end is marked with a quenching group BHQ3.

As a preferred embodiment, the probe operates at a concentration of 50-250nM, preferably 250nM.

The invention also provides a kit for detecting the Proteus, which comprises the primer probe combination.

As a preferred embodiment, the kit further comprises a digital PCR reaction buffer and nuclease-free water, wherein the digital PCR reaction buffer comprises one or more of TaqDNA polymerase, dNTPs, mg2+ and water.

The invention also provides a multiplex digital PCR detection method of the Proteus, which comprises the following steps:

1) Providing a sample nucleic acid to be tested;

2) Carrying out PCR amplification on the nucleic acid sample to be detected by adopting the primer probe combination to obtain a PCR product;

3) And collecting fluorescent signals of the PCR products and carrying out result quantification and judgment analysis.

As a preferred embodiment, the conditions for PCR amplification are: pre-denaturation at 95 ℃ for 5 min, 1 cycle; 2) Denaturation at 95℃for 30 seconds, annealing at 58℃for 45 seconds and extension for 45 cycles.

As a preferred embodiment, the decision analysis includes any one or more of the following:

(1) When the ATTO channel has a positive point, any one of the FAM, HEX, ROX, CY and CY5.5 channels has a positive point, and the copy number of the any one channel is close to that of the ATTO channel, judging that the sample to be tested has the Proteus corresponding to the any one channel only;

(2) When the ATTO channel has a positive point, any channel of FAM, HEX, ROX, CY and CY5.5 has a positive point, and the sum of the copy numbers of the positive channels except the ATTO channel is close to the copy number of the ATTO channel, judging that the detection contains all the Proteus corresponding to the channel with the positive point.

(3) And when the ATTO channel has no positive point and the other FAM, HEX, ROX, CY channels and CY5.5 channels have positive points, judging that the detection is abnormal, and retesting is needed.

The invention also provides application of the primer probe combination or the kit or the detection method in products for detecting the bacillus proteus for non-disease diagnosis and treatment.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention provides a primer probe capable of quantitatively detecting proteus, general proteus, proteus mirabilis, proteus penmansoni and proteus viscosus at the same time of high efficiency, sensitivity and specificity. Based on a dPCR platform, the reaction system is equally divided into tens of thousands of independent reaction units, so that the sensitivity of the reaction is greatly improved, the reaction can be fast (about 2 hours are consumed from the configuration of the reaction system to the analysis of the result), and meanwhile, the type and the number of the proteus in a sample to be detected can be absolutely quantified. By designing multiple TaqMan probes, the Proteus vulgaris, the Proteus mirabilis, the Proteus pensonii and the Proteus viscosus can be detected at the same time with high specificity, and the detection fixed value of multiple channels (belonging to and corresponding species) greatly improves the accuracy of the detection result during detection. The rapid detection of the bacillus proteus in urine, sputum and other samples is realized, the culture is not needed in the detection process, the detection period is short, the manpower is greatly reduced, and the detection efficiency is improved.

(2) The primer probe provided by the invention can accurately detect the content condition of the proteus in the sample to be detected under the extremely low copy number, and is more sensitive and accurate compared with other quantitative detection modes; the detection sensitivity is high, a standard curve is not required to be set, and the copy numbers of nucleic acids of the Proteus, the Proteus vulgaris, the Proteus mirabilis, the Proteus pensonii and the Proteus viscosus in the sample can be directly interpreted according to the detection result, so that the operation steps are greatly simplified; the method does not need to culture, reduces the workload of personnel and shortens the detection period, and is suitable for the rapid detection of clinical samples.

Drawings

Fig. 1: the digital PCR instrument result interpretation labeling example diagram in the embodiment 1 of the invention;

fig. 2: screening analysis chart of Proteus primer probe group in the embodiment 1 of the invention;

fig. 3: screening analysis chart of a common Proteus primer probe set in the embodiment 1 of the invention;

fig. 4: screening analysis chart of Proteus mirabilis primer probe group in the embodiment 1 of the invention;

fig. 5: screening analysis chart of the primer probe group of the Proteus pensonii in the embodiment 1 of the invention;

fig. 6: screening analysis chart of a primer probe group of Proteus hausei in the embodiment 1 of the invention;

fig. 7: screening analysis chart of a primer probe group of the Proteus viscosus in the embodiment 1 of the invention;

fig. 8: in the embodiment 1 of the invention, a screening analysis chart of a multi-primer probe group is provided; wherein, the ATTO channel is a Proteus detection channel, and other channels correspond to strain information: FAM channel corresponds to general Proteus, HEX channel corresponds to Proteus mirabilis, ROX channel corresponds to Proteus pensonii, CY5 channel corresponds to Proteus hawk, CY5.5 channel corresponds to Proteus viscosus.

Fig. 9: an analysis chart of the optimal working concentration of the Proteus probe in example 2 of the present invention; from left to right, the probe concentrations are 250nM, 200nM, 150nM, 100nM for the Proteus samples to be tested, and 250nM, 200nM, 150nM, 100nM for the deionized water as the negative control.

Fig. 10: an analysis chart of the optimal working concentration of the common Proteus probe in the embodiment 2 of the invention; from left to right, the probe concentrations are 250nM, 200nM, 150nM, 100nM groups for general Proteus as the sample to be tested, and 250nM, 200nM, 150nM, 100nM groups for deionized water as the negative control.

Fig. 11: an analysis chart of the optimal working concentration of the Proteus mirabilis probe in the embodiment 2 of the invention; from left to right, the probe concentrations are 250nM, 200nM, 150nM, 100nM groups for Proteus mirabilis as the sample to be tested, and 250nM, 200nM, 150nM, 100nM groups for deionized water as the negative control.

Fig. 12: an analysis chart of the optimal working concentration of the Proteus pensonii probe in example 2 of the present invention; from left to right, the probe concentrations were 250nM, 200nM, 150nM, 100nM for the sample to be tested and 250nM, 200nM, 150nM, 100nM for deionized water as the negative control.

Fig. 13: an analysis chart of the optimal working concentration of the Proteus hausei probe in the embodiment 2 of the invention; from left to right, the probe concentrations were 250nM, 200nM, 150nM, 100nM in the case of Proteus hausei as the sample to be tested, and 250nM, 200nM, 150nM, 100nM in the case of deionized water as the negative control.

Fig. 14: an analysis chart of the optimal working concentration of the Bacillus mucilaginosus probe in the embodiment 2 of the invention; from left to right, the probe concentrations were 250nM, 200nM, 150nM, 100nM for the A.viscosus as the sample to be tested, and 250nM, 200nM, 150nM, 100nM for the deionized water as the negative control.

Fig. 15: the detection result diagram of the probe specificity of the bacillus proteus primer in the embodiment 3 of the invention; eight chips from left to right, wherein the first two chips are a control group and are used for detecting a P.v and P.m mixed sample result graph; the third chip is a detection P.v result graph; the fourth chip is a detection P.m result diagram; the fifth chip is a detection P.p result graph; the sixth chip is a detection P.h result chart; the seventh chip is a result diagram for detecting P.my; the eighth chip was a negative control.

Fig. 16: in the embodiment 3 of the invention, a probe specificity result diagram of a primer for detecting the bacillus proteus by using common pathogenic bacteria is sequentially as follows from left to right: escherichia coli, klebsiella pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii, streptococcus pneumoniae, salmonella, neisseria gonorrhoeae, haemophilus influenzae, and negative control group.

Fig. 17: the positive control detection result diagram of the common pathogenic bacteria in the embodiment 3 of the invention is detected by using a primer probe designed in a laboratory to assist in proving the specificity of the primer probe of the proteus, wherein the primer probe of the proteus comprises the following components in sequence from left to right: escherichia coli, klebsiella pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii, streptococcus pneumoniae, salmonella, neisseria gonorrhoeae, and Haemophilus influenzae.

Fig. 18: the sensitivity detection result diagram of Proteus in the embodiment 4 of the invention; from left to right, two chips each for detecting Proteus vulgaris, proteus mirabilis, proteus pensonii, proteus hausensis and Proteus viscosus and 1 negative control are shown, respectively.

Fig. 19: the sensitivity detection result diagram of the general Proteus in the embodiment 4 of the invention; 10 parallel tests and 1 negative control of the primer probe group of Proteus vulgaris are shown in sequence from left to right.

Fig. 20: the sensitivity detection result diagram of the Proteus mirabilis in the embodiment 4 of the invention; 10 parallel tests and 1 negative control of the primer probe set of Proteus mirabilis are shown in sequence from left to right.

Fig. 21: the sensitivity detection result diagram of the Proteus pensonii in the embodiment 4 of the invention; 10 parallel tests and 1 negative control of the primer probe set of Proteus pensonii are shown in sequence from left to right.

Fig. 22: the sensitivity detection result diagram of the Proteus hausei in the embodiment 4 of the invention; 10 parallel tests and 1 negative control of the primer probe group of Proteus hao are shown in sequence from left to right.

Fig. 23: the sensitivity detection result diagram of the Proteus viscosus in the embodiment 4 of the invention; 10 parallel tests and 1 negative control of the primer probe set for producing Proteus viscosus are shown in sequence from left to right.

Fig. 24: an actual sample detection result diagram in the embodiment 5 of the invention; wherein, two repeated chips of samples 1 and 2 to be tested and 1 negative control are sequentially arranged from left to right.

Detailed Description

In order to more clearly describe the technical scheme and optimization steps of the present invention, the present invention will be described in further detail in conjunction with the following specific embodiments, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, modifications, equivalents, improvements, etc., which are apparent to those skilled in the art without the benefit of this disclosure, are intended to be included within the scope of this invention.

Example 1: screening of primer probe combinations

2 sets of primers are designed on the Proteus atpD gene for amplification, and 6 probes are designed at different positions for detecting Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii, proteus hausensis and Proteus viscosus respectively. Primer probe sequence information and corresponding numbers are shown in table 1:

TABLE 1

Wherein the probe of Proteus is labeled with a fluorescent group ATTO425 at the 5 'end and a quenching group BHQ1 at the 3' end;

the 5 'end of the probe of the general Proteus is marked with a fluorescent group FAM, and the 3' end is marked with a quenching group MGB;

labeling a fluorescent group HEX at the 5 'end and a quenching group MGB at the 3' end of a probe of Proteus mirabilis;

marking a fluorescent group ROX at the 5 'end of a probe of the Proteus pensonii and marking a quenching group BHQ2 at the 3' end;

marking a fluorescent group CY5 at the 5 'end and a quenching group BHQ2 at the 3' end of a probe of the Proteus delbrueckii;

the 5 'end of the probe of the Proteus viscosus is marked with a fluorescent group CY5.5, and the 3' end is marked with a quenching group BHQ3.

1.1 screening of Proteus primer probe combinations

The above Proteus primer probes in Table 1 were combined into combination 1 and combination 2, respectively, as shown in Table 2:

TABLE 2

1.2 screening of Proteus vulgaris primer probe combinations

The general Proteus primer probes in Table 1 were combined into combination 1 and combination 2, respectively, as shown in Table 3:

TABLE 3 Table 3

1.3 screening of Proteus mirabilis primer probe combinations

The Proteus mirabilis primer probes in Table 1 were combined into combination 1 and combination 2, respectively, as shown in Table 4:

TABLE 4 Table 4

1.4 screening of Proteus pensonii primer probe combinations

The primer probes of Proteus pensonii in Table 1 were combined into combination 1 and combination 2, respectively, as shown in Table 5:

TABLE 5

1.5 screening of Proteus hausei primer probe combinations

The above-mentioned Proteus hausei primer probes in Table 1 were combined into combination 1 and combination 2, respectively, as shown in Table 6:

TABLE 6

1.6 screening of Proteus viscosus primer probe combinations

The above-mentioned Proteus viscosus primer probes in Table 1 were combined into combination 1 and combination 2, respectively, as shown in Table 7:

TABLE 7

1.7 multiplex Proteus primer probe combination screening

The primer probes of Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii, proteus haos and Proteus viscosus in Table 1 were combined into combinations 1 and 2, respectively, as shown in Table 8:

TABLE 8

1.8PCR reaction

(1) Obtaining nucleic acid to be tested, purchasing strain glycerinum and 5ug DNA in Testethoscopic biological network (https:// testobio.com /), general bacillus proteus (TS 274748/ATCC 6896) and Proteus mirabilis (TS 208253/ATCC 15146), and other 3 strains (Proteus pensonii, proteus hausei and Proteus viscosus) for laboratory preservation.

(2) Reaction buffer (dPCR Mix), precious quasi-organism dPCR premix (10×), cat No.: MX0101, composition: taq enzyme, UDG enzyme, dNTPs, mg ²⁺ Tween and ROX dye.

(3) The reaction system configuration is shown in table 9:

TABLE 9

Reagent(s)	Working volume
		dPCR premix (10×)	2uL
Primer probe Mix (20×)	1uL
		Sample DNA	≥2uL
Nuclease-free water	Complement 20uL

(4) Reaction conditions:

1) Pre-denaturation at 95 ℃ for 5 min, 1 cycle;

2) Denaturation at 95℃for 30 seconds, annealing at 58℃for 45 seconds and extension for 45 cycles.

1.9 digital PCR workflow

A digital Amp digital PCR system manufactured by Shanghai, inc. was used, and the digital PCR system comprises a digital PCR sample preparation instrument (INLD 100A), a gene amplification instrument (INAP 100B), and a digital PCR reader (INRD 206A).

(1) Sample adding: the reaction system was configured according to step 1.8 (3), and the sample was loaded using a digital PCR sample preparation instrument (INLD 100A), and 20uL of the reaction system was evenly distributed to 2-ten thousand well chips.

(2) Amplification: the prepared chip was placed on a gene amplification apparatus (INAP 100B) and amplified under the reaction conditions of step 1.8 (4).

Note that: the PCR reaction liquid with the sample is added or the chip with the sample is put on the machine immediately for experiment; if the on-line reaction cannot be performed in time due to special reasons, the PCR reaction solution or the chip after sample introduction should be stored at 4 ℃ for a storage time of not more than 12 hours.

(3) Chip reading analysis: the amplified chip was placed in a digital PCR reader (INRD 206A) for chip reading analysis.

Result judgment criteria: the reading result is presented as a two-dimensional scatter diagram, and as shown in fig. 1, the upper shadow part is a positive point gray value (brightness), and the bottom shadow part is a negative point gray value (brightness); the positive point has high gray value, and the negative point has low gray value, namely the gap value between the positive point and the negative point has good effect. Under the condition that the negative gray values are consistent, the larger the positive point gray value is, the larger the gap value between the positive point and the negative point is, which shows that the effect is better.

And (3) setting: the instrument system calculates the actual concentration of the sample according to the Poisson distribution of the statistical formula, and outputs the result by taking the copies/uL as a unit.

And (3) quality control: the ATTO channel is a detection channel of Proteus, and other channels correspond to strain information: FAM channel corresponds to general Proteus, HEX channel corresponds to Proteus mirabilis, ROX channel corresponds to Proteus pensonii, CY5 channel corresponds to Proteus hawk, CY5.5 channel corresponds to Proteus viscosus. Therefore, when any Proteus is detected, ATTO and the corresponding strain channel have positive points.

Note that: when oil or water is present in the chip reading frame, the chip reading frame can be wiped by dust-free cloth.

Interpretation of the test results:

(1) The ATTO channel has a positive point, any one of the channels FAM, HEX, ROX, CY and CY5.5 has a positive point, the channel is close to the ATTO channel in copy number, and the test sample has and only has the Proteus corresponding to the channel;

(2) The ATTO channel has positive points, any channel of FAM, HEX, ROX, CY and CY5.5 has positive points, and the sum of the copy numbers of the positive channels except the ATTO channel is close to the copy number of the ATTO channel, so that the reaction contains all the proteus corresponding to the channel with the positive points;

(3) ATTO channels have no positive points, and the other 5 channels have positive points, so that the test is abnormal and the test needs to be retested.

1.10 screening results of primer probe combinations

The test result of the combination of the primer probes of the Proteus is shown in figure 2, and the corresponding combination 2 is selected;

the test result of the common Proteus primer probe combination is shown in figure 3, and the corresponding combination 2 is selected;

the test result of the combination of the primer and the probe of the Proteus mirabilis is shown in figure 4, and the corresponding combination 2 is selected;

the result of the combination test of the primer probe of the Proteus pensonii is shown in figure 5, and the corresponding combination 2 is selected;

the test result of the combination of the primer and the probe of the Proteus hao is shown in figure 6, and the corresponding combination 2 is selected;

the test result of the primer probe combination of the Proteus viscosus is shown in figure 7, and the corresponding combination 2 is selected;

the test result of the multiplex Proteus primer probe combination is shown in figure 8, and the corresponding combination 2 is selected;

example 2: screening of probe working concentrations of different Proteus species

In this example, the above 6 probes were subjected to working concentration screening according to primer probe combinations 2 of each of the genus Bacillus and 5 species thereof determined in the above example 1, and each probe was subjected to digital PCR test with 4 gradients (100, 150, 200, 250 nM).

FIGS. 9 to 14 show the results of the probe concentration gradient test of Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii, proteus hausensis, proteus viscosus, respectively, wherein eight chips in each of the figures represent, from left to right, the probe concentration of 250nM, 200nM, 150nM, 100nM groups when each of the above-mentioned bacteria was used as a sample to be tested, and the probe concentration of 250nM, 200nM, 150nM, 100nM groups when deionized water was used as a negative control, respectively; as can be seen from the results of FIGS. 9 to 14, the gap value between the positive and negative spots is maximized according to the instrument interpretation standard at a probe working concentration of 250nM for each test bacterium. And finally, the working concentration of the probes of the Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii, proteus hausei and Proteus viscosus is 250nM.

Example 3: proteus detection kit specificity test

In this example, a specificity test was performed according to the primer probe combinations and the optimal working concentrations of the probes determined in example 1 and example 2. The test contents are as follows:

(1) Detecting the other 5 Proteus probe specificities by using general Proteus vulgaris nucleic acid (P.v) nucleic acid;

(2) Other 5 Proteus probes were tested for specificity using Proteus mirabilis (Proteus mirabilis, P.m);

(3) Other 5 Proteus probes were tested for specificity using Proteus penneri (P.p);

(4) Other 5 Proteus probes were tested for specificity using Proteus hauseri (P.h);

(5) Other 5 Proteus probe specificities were detected using Proteus viscosus (Proteus myxofaciens, P.my);

(6) The primer probe specificity of Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii, proteus hausei and Proteus viscosus was detected by using some common pathogenic bacteria (9). Common pathogenic bacteria: escherichia coli, klebsiella pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii, streptococcus pneumoniae, salmonella, neisseria gonorrhoeae and Haemophilus influenzae, all of which are purchased from Testosterone organisms, and primer probes designed for the common pathogens are simultaneously used for detection and positive control.

The specific implementation steps are as follows:

the samples to be tested were subjected to 14 reactions in total, 2 reactions were additionally set as controls (nucleic acid samples containing general Proteus and Proteus mirabilis), 16 reactions were added in total, and in a reaction system in which 2uL to 20uL of sample DNA was added for each reaction, 18 parts of sample reaction liquid was not added according to 1.8 (3) in example 1, 18uL of reaction system was taken and packed into 16 Ep tubes, and 2uL of nucleic acid samples were added respectively, and dPCR detection was performed according to the digital PCR workflow of 1.9.

FIG. 15 is a diagram showing the results of detection of the probe specificity of the Proteus primer of the present invention, wherein eight chips from left to right, the first two chips being a control group, are used for detecting a mixed sample of P.v and P.m; the third chip is a detection P.v result graph; the fourth chip is a detection P.m result diagram; the fifth chip is a detection P.p result graph; the sixth chip is a detection P.h result chart; the seventh chip is a result diagram for detecting P.my; the eighth chip was a negative control.

FIG. 16 is a graph showing the results of detection of Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii, proteus hausei and Proteus viscosus primer probe specificities by using common pathogenic bacteria, which are shown in the following order from left to right: escherichia coli, klebsiella pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii, streptococcus pneumoniae, salmonella, neisseria gonorrhoeae, haemophilus influenzae, and negative control group. Fig. 17 is a graph of the positive control detection result of the above common pathogenic bacteria, wherein the following steps are sequentially performed from left to right: escherichia coli, klebsiella pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii, streptococcus pneumoniae, salmonella, neisseria gonorrhoeae, and Haemophilus influenzae.

Combining the experimental results of fig. 15-17, the results of this example demonstrate that: the invention detects the primer probe group of the bacillus proteus:

1. in detection P.v, only the ATTO channel has a positive spot with the FAM channel;

2. at the time of detection P.m, only ATTO channels had positive spots with HEX channels;

3. at the time of detection P.p, only ATTO channels had positive spots with ROX channels;

4. in the detection of P.h, only ATTO channels were positively dotted with CY5 channels;

5. in the detection of p.my, only ATTO channels were positively dotted with CY5.5 channels;

6. when detecting common pathogens, the positive control detection is normal, and the primer probes have no positive result.

The detection results of this example 3 were combined to demonstrate that the primer probe set for detecting Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii, proteus hausen and Proteus viscosus of the present invention has strong specificity.

Example 4: proteus detection kit sensitivity test

In this example, a sensitivity test was performed according to the primer probe combinations and the optimal working concentrations of the probes determined in example 1 and example 2.

The specific test contents are as follows: the nucleic acids of the general proteus, the proteus mirabilis, the proteus penmansoni, the proteus haemanensis and the proteus viscosus with the concentration of about 50copies/uL are diluted by 100 times, and the final concentration is controlled to be less than the quantitative detection limit (1 copies/uL) of the precious biological digital PCR platform. The reaction system was configured according to 1.8 (3) in example 1, and 10 parallel tests and 1 negative control were performed for each primer probe set, and digital PCR was performed according to 1.9 in example 1, and the detection results are shown in FIGS. 18 to 23, respectively:

FIG. 18 shows graphs of results of detection limit dilution of Proteus vulgaris, proteus mirabilis, proteus penmansoni, proteus hausensis and Proteus viscosus using Proteus primer probe set, wherein two chips and 1 negative control for detection of Proteus vulgaris, proteus mirabilis, proteus penmansoni, proteus hausensis and Proteus viscosus respectively from left to right, detection rate 100%;

FIG. 19 shows a graph of results of Proteus vulgaris after dilution with detection limits of Proteus vulgaris primer probe sets, wherein 10 parallel test sets for detecting Proteus vulgaris and 1 negative control set are shown from left to right, respectively; the detection rate is 100 percent.

FIG. 20 shows a graph of Proteus mirabilis results after limiting dilution with Proteus mirabilis primer probe set, wherein 10 parallel test sets for detecting Proteus mirabilis and 1 negative control set are shown from left to right, respectively; the detection rate is 100 percent.

FIG. 21 shows graphs of the results of Proteus penmansoni after dilution of the detection limit using the Proteus penmansoni primer set, wherein 10 parallel test sets for detection of Proteus penmansoni and 1 negative control set are shown from left to right, respectively; the detection rate is 100 percent.

FIG. 22 shows a graph of the results of Proteus hausei after dilution with the detection limit of Proteus hausei primer probe set, wherein 10 parallel test sets for detecting Proteus hausei and 1 negative control set are shown from left to right, respectively; the detection rate is 100 percent.

FIG. 23 shows graphs of the results of Proteus viscosus after limiting dilution with the Proteus viscosus primer probe set, wherein 10 parallel test sets for detecting Proteus viscosus and 1 negative control set are shown from left to right, respectively; the detection rate is 100 percent.

The results of this example 4 show that the detection limit of the primer probe set of the kit for detecting Proteus of the present invention is not higher than 1.0copies/uL, which indicates that the kit has high sensitivity and can perform normal detection even for samples with very small template content.

Example 5: detection of the primer probe combination of the invention on an actual sample

In this example, detection of an actual sample was performed according to the primer probe combinations and the optimal working concentrations of the probes determined in examples 1 and 2.

The specific detection content is as follows:

(1) 2 samples to be tested, wherein each sample is subjected to 1 parallel test, and the total number of the test results is 4;

(2) Extracting nucleic acid of a sample to be detected by using a full-gold automatic extractor (TS-32);

(3) The reaction system was configured as in 1.8 (3) of example 1, with an addition amount of 5uL per reaction sample;

(4) The dPCR assay was performed as in example 1, 1.9.

Fig. 24 is a diagram of the detection result of detecting an actual sample by using the primer probe of the present invention, wherein two repeated chips of samples 1 and 2 to be detected and 1 negative control are sequentially arranged from left to right. As shown in FIG. 24, the Proteus detection kit provided by the invention can normally detect the Proteus in samples 1 and 2 to be detected, wherein the sample 1 to be detected shows that the concentration of the common Proteus is 902.87copies/uL and the concentration of the Proteus mirabilis is 1300.01copies/uL; sample 2 to be tested showed that the general Proteus concentration 491.74copies/uL and the Proteus mirabilis concentration 657.19copies/uL were detected, and no other Proteus was detected since the sum of the general Proteus and Proteus mirabilis detected by both samples was substantially identical to the copy number detected by the Proteus channel.

The foregoing is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications are within the scope of the present invention.

Claims

1. A primer probe combination for multiplex digital PCR detection of proteus, characterized in that the primer probe combination is used for detecting proteus genus and simultaneously distinguishing general proteus and/or proteus mirabilis and/or proteus pensis and/or proteus haelii and/or proteus viscosus, and the nucleotide sequence of the primer is as follows:

SEQ ID NO：2 5’-ATCATGAACGTTCTGGGTACACC-3’

SEQ ID NO：4 5’-GATCGAGCACTCAGGTTACTCTG-3’。

2. the primer probe combination of claim 1, wherein the nucleotide sequence of the probe comprises:

the nucleotide sequence of the probe of the Proteus is shown as SEQ ID NO:5 is shown in the figure;

the nucleotide sequence of the probe of the general Proteus is shown as SEQ ID NO:6 is shown in the figure;

the nucleotide sequence of the Proteus mirabilis probe is shown in SEQ ID NO: shown in figure 7;

the nucleotide sequence of the probe of the Proteus pensonii is shown as SEQ ID NO: shown as 8;

the nucleotide sequence of the probe of the Proteus hao is shown as SEQ ID NO: shown as 9;

the nucleotide sequence of the probe of the Proteus viscosus is shown as SEQ ID NO: shown at 10.

3. The primer probe combination of claim 2, wherein the probes are labeled with a fluorescent group and a quenching group, respectively, the fluorescent group being selected from any one of ATTO, FAM, HEX, ROX, CY or CY 5.5; the quenching group is selected from any one of BHQ1, BHQ2, BHQ3 or MGB, and the fluorescent groups selected from Proteus, proteus vulgaris, proteus mirabilis, proteus pensonii and Proteus viscosus are different.

4. A primer probe combination according to claim 3, wherein when the quenching group is selected from MGBs, the fluorescent group is preferably FAM or HEX.

5. The primer probe combination of claim 2, wherein the probe of the genus proteus is labeled with a fluorescent group ATTO at the 5 'end and a quenching group BHQ1 at the 3' end;

the 5 'end of the probe of the Proteus mirabilis is marked with a fluorescent group HEX, and the 3' end is marked with a quenching group MGB;

the 5 'end of the probe of the Proteus pensonii is marked with a fluorescent group ROX, and the 3' end is marked with a quenching group BHQ2;

the 5 'end of the probe of the Haofacillus is marked with a fluorescent group CY5, and the 3' end is marked with a quenching group BHQ2;

6. The primer probe combination of any one of claims 1 to 5, wherein the working concentration of the probe is 50-250nM, preferably 250nM.

7. A kit for detection of proteus comprising the primer probe combination of any one of claims 1-6.

8. The kit of claim 7, further comprising a digital PCR reaction buffer and water free of ribozymes, wherein the digital PCR reaction buffer comprises TaqDNA polymerase, dNTPs, mg ²⁺ And one or more of water.

9. A multiplex digital PCR detection method of Proteus comprises the following steps:

1) Providing a sample nucleic acid to be tested;

2) Performing PCR amplification on the nucleic acid sample to be detected by using the primer probe combination according to any one of claims 1-6 to obtain a PCR product;

10. The method according to claim 9, wherein the conditions for PCR amplification are: pre-denaturation at 95 ℃ for 5 min, 1 cycle; 2) Denaturation at 95℃for 30 seconds, annealing at 58℃for 45 seconds and extension for 45 cycles.

11. The method of claim 9, wherein the decision analysis includes any one or more of:

1) When the ATTO channel has a positive point, any one of the FAM, HEX, ROX, CY and CY5.5 channels has a positive point, and the copy number of the any one channel is close to that of the ATTO channel, judging that the sample to be tested has the Proteus corresponding to the any one channel only;

2) When the ATTO channel has positive points, any channel of FAM, HEX, ROX, CY and CY5.5 has positive points, and the sum of the copy numbers of the positive channels except the ATTO channel is close to the copy number of the ATTO channel, judging that the detection contains all the Proteus corresponding to the channels with positive points;

3) And when the ATTO channel has no positive point and the other FAM, HEX, ROX, CY channels and CY5.5 channels have positive points, judging that the detection is abnormal, and retesting is needed.

12. Use of a primer probe combination according to any one of claims 1-6 or a kit according to any one of claims 7-8 or a detection method according to claims 9-11 in a product for detection of proteus for non-disease diagnostic and therapeutic purposes.