CN106609303B - Primer and method for detecting enterobacter cloacae O20 type - Google Patents

Primer and method for detecting enterobacter cloacae O20 type Download PDF

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CN106609303B
CN106609303B CN201510708360.1A CN201510708360A CN106609303B CN 106609303 B CN106609303 B CN 106609303B CN 201510708360 A CN201510708360 A CN 201510708360A CN 106609303 B CN106609303 B CN 106609303B
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enterobacter cloacae
nucleotide sequences
primer
primers
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李雅玥
王晓彤
李彤
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Tianjin Third Central Hospital
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Abstract

The invention discloses a primer and a method for detecting Enterobacter cloacae O20 type, wherein the primer is one of two groups of nucleotide sequences, and the forward primer of the first group of nucleotide sequence is 5 '-AGAATCTTACACTGGCTTA-3'; reverse primers 5 '-CTTAATACACCTGGCACA-3'; forward primers 5 '-TTTGGCTATGGCCGTATT-3' of a second set of nucleotide sequences; the reverse primers 5 '-GTGCAGATGCCTGATGAA-3'. During detection, extracting DNA of a sample to be detected, performing PCR amplification by using the first group of nucleotide sequences or the second nucleotide sequences as primers, and performing electrophoresis detection on an amplification product. The invention avoids the defects of the traditional identification method, and has short time, strong specificity and high sensitivity for detecting the enterobacter cloacae O20.

Description

Primer and method for detecting enterobacter cloacae O20 type
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer and a method for detecting Enterobacter cloacae O20.
Background
Enterobacter cloacae (Enterobacter cloacae) is a gram-negative thick short bacillus, widely exists in nature, can be detected in soil, plants, insects and human and animal feces, is one of normal strains in human and animal intestinal tracts, and is also an important conditional pathogen. In recent years, due to the wide clinical use of third and fourth generation cephalosporins, carbapenems and other ultra-broad spectrum antibacterial drugs, the number of drug-resistant strains of the bacteria is increasing, so that the clinical treatment of infectious diseases caused by the bacteria tends to be complicated. The main reason for the emergence of resistant strains is the failure to identify the pathogens at the first time and, in the case of unidentified pathogens, the misuse of drugs. Since the 80 s, means for serotype typing, phage typing, bacteriocin typing, biotyping, and the like of Enterobacter cloacae have been reported in the literature. However, the above typing methods can only be used in combination, and since the reproducibility of typing by using one method alone is not good and the typing ability is weak, the accurate typing of Enterobacter cloacae cannot be well performed. Therefore, a rapid and accurate detection means for identifying pathogens is urgently needed clinically, so that targeted treatment can be performed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a detection primer and a detection method for enterobacter cloacae type O20. The method can be used for PCR detection of enterobacter cloacae O20 type, and has the advantages of short detection time, low cost, high specificity of detection result, easy judgment of result and strong practicability.
The technical object of the present invention is achieved by the following technical means.
A primer for detecting Enterobacter cloacae O20 type is one of the following two groups of nucleotide sequences, wherein each group of nucleotide sequence consists of a forward primer and a reverse primer:
a first set of nucleotide sequences: forward primers (SEQ ID NO.1)5 '-AGAATCTTACACTGGCTTA-3'; reverse primers (SEQ ID NO.2)5 '-CTTAATACACCTGGCACA-3';
a second set of nucleotide sequences: forward primers (SEQ ID NO.3)5 '-TTTGGCTATGGCCGTATT-3'; the reverse primers (SEQ ID NO.4)5 '-GTGCAGATGCCTGATGAA-3'.
The application of the primers in detecting the enterobacter cloacae O20 type, namely the method for detecting the enterobacter cloacae O20 type, comprises the steps of firstly extracting DNA of a sample to be detected, taking the sample DNA as a template, carrying out PCR amplification by using a first group of nucleotide sequences or a second nucleotide sequence as a primer, then carrying out agarose gel electrophoresis detection on a PCR amplification product, and judging whether the sample contains the enterobacter cloacae O20 type.
In the detection method, when the first group of nucleotide sequences are used as primers, if a single band appears at a 459bp position in an electrophoresis result, the sample contains the Enterobacter cloacae O20 type; on the other hand, the sample does not contain Enterobacter cloacae type O20.
In the above detection method, when the second set of nucleotide sequences is used as primers, if a single band appears at the position of 246bp as a result of electrophoresis, the sample contains Enterobacter cloacae type O20; on the other hand, the sample does not contain Enterobacter cloacae type O20.
In the above detection method, the reaction system for performing PCR amplification is a 30. mu.L reaction system, specifically: 10 XPCR buffer (containing Mg)2+)2.5 mu L; 2.5mmol/L dNTP 1 μ L; 1.5-2.25U of Taq enzyme; 0.1-0.3. mu.L of 10. mu. mol/L of a primer (i.e., a primer for detecting Enterobacter cloacae type O20, a primer pair represented by the first set of nucleotide sequences or a primer pair represented by the second set of nucleotide sequences); 1. mu.L of sample DNA (i.e., template DNA) at 10-300 ng/. mu.L; finally, 30. mu.L of the solution was made up with sterilized ultrapure water.
In the above detection method, when PCR amplification is performed, the PCR reaction procedure is: 5min at 95 ℃ for 1 cycle; at 94 ℃ for 30s, at 58-65 ℃ for 45s and at 68 ℃ for 1min, for 35 cycles; 7min at 72 ℃ for 1 cycle; storing at 4 ℃.
In the detection method, 10 μ L of PCR amplification product is subjected to 1% agarose gel electrophoresis detection, and sampling detection is performed from the PCR amplification product during detection, namely the reaction system of PCR amplification is a PCR detection system.
Compared with the prior art, the invention has the following advantages: the detection method of the invention has the advantages of short time, strong specificity and high sensitivity for detecting the Enterobacter cloacae O20. The method avoids the defects of complex operation, long time consumption, low accuracy, low detection rate and the like of the traditional identification method. Meanwhile, instruments, reagents and the like related to the invention are common, detection work can be carried out in a common basic laboratory, and the practicability is higher. The target spot detected by the invention has the advantages of singleness, specific detection result, easy judgment, short required time, detection cost reduction and detection efficiency improvement.
Drawings
FIG. 1 is a diagram showing the results of gel electrophoresis in the PCR detection method of the example of specificity evaluation experiment.
FIG. 2 is a diagram showing the results of gel electrophoresis in the test for evaluating sensitivity of the PCR detection method in the examples.
FIG. 3 is a diagram showing the results of gel electrophoresis in clinical sample detection in the examples.
Detailed Description
The technical scheme of the invention is further explained by combining specific examples. The specific conditions not specified in the examples are generally carried out according to conventional conditions such as those described in molecular cloning, Sambrook et al, a Laboratory Manual (New York: Cold spring Habor Laboratory Press,1989), or according to the manufacturer's recommendations.
Experimental reagent
Distilled water Milli-Q System, Millipore Co., USA
10 XPCR Buffer (containing Mg)2+) Bao bioengineering (Dalian) Co Ltd
2.5mmol/L dNTP Bao bioengineering (Dalian) Co Ltd
Taq enzyme Bao bioengineering (Dalian) Co Ltd
DL1000 molecular weight Standard Bao bioengineering (Dalian) Co Ltd
100bp DNA Ladder molecular weight standard Bao bioengineering (Dalian) Co Ltd
1% agarose gel Genview Co.,USA
Laboratory apparatus
Constant temperature shaking table Shanghai-Ping laboratory Equipment Ltd
Spectrophotometer Eppendorf, BioPhotometer, Germany
Gel imaging system Shanghai Tanon corporation, GIS 2010
Ultrapure water instrument Millipore Co.,USA
Centrifugal machine Hunan instrument laboratory Instrument development Co., Ltd, H1650-W
Experimental strains
Name of bacterium Origin of origin Original numbering of strains
Enterobacter cloacae O20 strain NCTC 11589
Escherichia coli O22 CMCC 44182
Salmonella typhi O67 strain CMCC 50071-9
Shigella dysenteriae O1 strain CMCC 51491
Shigella boydii powdai O2 strain CMCC C 2
CMCC: china medical microorganism strain preservation management center, namely China medical bacteria preservation management center, Beijing City eastern region Tiantan Xili No. 2; NCTC: national Collection of Type Cultures, Central public health Laboratory, London, United Kingdom National Standard Collection of strains.
Step one, a specific primer pair for enterobacter cloacae type O20 was designed and synthesized by Invitrogen fundic fundi (shanghai) trade ltd.
A first set of nucleotide sequences: forward primers 5 '-AGAATCTTACACTGGCTTA-3'; reverse primers 5 '-CTTAATACACCTGGCACA-3'; a second set of nucleotide sequences: forward primers 5 '-TTTGGCTATGGCCGTATT-3'; the reverse primers 5 '-GTGCAGATGCCTGATGAA-3'. Step two, DNA template preparation
Inoculating Enterobacter cloacae O20 in 5mL nutrient broth liquid culture medium (see J.Sambrook et al, introduction to molecular cloning, second edition) 909 page), shake-culturing in a 35 ℃ constant temperature shaking table for 12h to increase bacteria, taking 1mL of bacterial liquid, and putting into 1.5mL of sterile centrifuge tube; centrifuging at 12000r/min for 15min, discarding supernatant, adding 500 μ L sterilized ultrapure water, gently blowing and beating with pipette, re-suspending thallus, centrifuging at 12000r/min for 15min, discarding supernatant, and collecting bacteria; adding 100 μ L sterilized ultrapure water, gently blowing with a pipette, resuspending the thallus, boiling in boiling water for 15min, immediately taking out, and standing at-20 deg.C for 30 min. Then unfreezing at 35 ℃, centrifuging at 12000r/min for 15min, and taking supernatant and placing at 4 ℃ for later use or storing at-20 ℃.
Step three, establishing a specific PCR detection method for enterobacter cloacae O20
The PCR detection system is 30 mu L (namely a reaction system for carrying out PCR amplification), and the reaction system specifically comprises: 10 XPCR buffer (containing Mg)2+) 2.5. mu.L, 1. mu.L of 2.5mmol/L dNTP, 1.5U of Taq enzyme, 0.3. mu.L of 10. mu. mol/L primer (first set of nucleotide sequences or second set of nucleotide sequences), 1. mu.L of 100 ng/. mu.L template DNA (prepared in step 2), and finally supplementing to 30. mu.L with sterilized ultrapure water; the PCR detection reaction program is as follows: 5min at 95 ℃ for 1 cycle; at 94 ℃ for 30s, at 60 ℃ for 45s and at 68 ℃ for 1min, for 35 cycles; 7min at 72 ℃ for 1 cycle; storing at 4 ℃.
Step four, judging the PCR amplification result by gel electrophoresis
The judging method specifically comprises the following steps: taking 10 mu L of PCR amplification product, detecting by 1% agarose gel electrophoresis, and observing the electrophoresis result under the irradiation of an ultraviolet lamp:
when the first group of nucleotide sequences are used as primers, if a single band appears at a 459bp position in an electrophoresis result, the sample contains the enterobacter cloacae O20 type; on the other hand, the sample does not contain Enterobacter cloacae type O20.
When the second group of nucleotide sequences are used as primers, if a single band appears at the position of 246bp as a result of electrophoresis, the sample contains the Enterobacter cloacae O20 type; on the other hand, the sample does not contain Enterobacter cloacae type O20.
PCR detection method specificity evaluation experiment
According to the DNA template preparation and PCR detection method, the stored enterobacter cloacae, escherichia coli, salmonella, shigella dysenteriae and shigella boydii are subjected to PCR amplification reaction, the amplified product is subjected to 1% agarose gel electrophoresis detection, and the specificity detection result is shown in figure 1.
In the figure: lane M is a 100bp DNA Ladder molecular weight standard; lanes 1 and 7 are both negative controls with template sterilized ultrapure water; the first group of nucleotide sequences are adopted as primers, the detection results are shown in lanes 2-6, and respectively comprise Enterobacter cloacae O20 strain of Enterobacter cloacae, Escherichia coli O22 strain of Escherichia coli, Salmonella typhi O67 strain, Shigella dysenteriae O1 strain, Shigella bodydiiO 2 strain of Botrytis baumannii, only Enterobacter cloacae O20 strain has a specific band at 459bp, and other strains do not have specific bands; the second set of nucleotide sequences was used as primers, and the detection results are shown in lanes 8-12, which are Enterobacter cloacae O20 strain of Enterobacter cloacae, Escherichia coli O22 strain of Escherichia coli, Salmonella Salmonella typhi O67 strain, Shigella dysenteriae O1 strain, Shigella bodydii O2 strain of Shigella bodydii, and specific bands appeared only in 246bp of Enterobacter cloacae O20 strain, but not in other strains.
PCR detection method sensitivity evaluation experiment
The sensitivity of the PCR detection method is indicated by the lowest DNA mass that can be detected in each reaction (i.e., the mass of sample DNA in the reaction system after PCR amplification is performed). The genomic DNA extract of the pure culture was sampled, the concentration of the DNA was measured with a spectrophotometer, and gradient dilution (concentration dilution) was performed with different fold using sterilized ultrapure water to obtain gradient DNA (Enterobacter cloacae O20 strain) of 1000 ng/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, and 1 ng/. mu.L, and the above-mentioned gradient DNA was subjected to PCR amplification under the same conditions (PCR amplification was performed using 1. mu.L of the gradient dilution as a template), and the amplification product was detected by gel electrophoresis, and the result of gel electrophoresis was observed under irradiation of an ultraviolet lamp, as shown in FIG. 2. In fig. 2: lane M is DL1000 molecular weight standard; lanes 1-4 show the PCR amplification results of Enterobacter cloacae O20 templates at 1000ng, 100ng, 10ng, and 1ng, using the first set of nucleotide sequences as primers; lanes 5-8 show the results of PCR amplification of Enterobacter cloacae O20 templates at 1000ng, 100ng, 10ng, and 1ng using the second set of nucleotide sequences as primers. As can be seen from FIG. 2, no matter the first set of nucleotide sequences or the second set of nucleotide sequences of the present invention are used, the lowest amount of DNA template detectable by the detection method is 10ng, and the method has good sensitivity.
Clinical suspected strain detection
2 suspected strains clinically isolated were detected by the enterobacter cloacae O20-type specific PCR detection method established as described above. The detection result is shown in FIG. 3, wherein the lane M is a 100bp DNA Ladder molecular weight standard; lane 1 is a positive control of Enterobacter cloacae type O20, lanes 2-3 are 2 suspected strains, lane 4 is a negative control of sterilized ultrapure water as a template, and lanes 1-4 all use the first set of nucleotide sequences as primers; lane 5 is a positive control of Enterobacter cloacae type O20, lanes 6-7 are 2 suspected strains, lane 8 is a negative control with a template of sterilized ultrapure water, and lanes 5-8 all use a second set of nucleotide sequences as primers. As can be seen, 2 clinically suspected strains showed negative results.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
Figure IDA0000831738420000011
Figure IDA0000831738420000021

Claims (1)

1. A primer for detecting Enterobacter cloacae type O20, wherein the primer is one of the following two groups of nucleotide sequences, and each group of nucleotide sequence consists of a forward primer and a reverse primer: a first set of nucleotide sequences: forward primers 5 '-AGAATCTTACACTGGCTTA-3'; reverse primers 5 '-CTTAATACACCTGGCACA-3'; a second set of nucleotide sequences: forward primers 5 '-TTTGGCTATGGCCGTATT-3'; the reverse primers 5 '-GTGCAGATGCCTGATGAA-3'.
CN201510708360.1A 2015-10-27 2015-10-27 Primer and method for detecting enterobacter cloacae O20 type Expired - Fee Related CN106609303B (en)

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CN102952881A (en) * 2012-10-25 2013-03-06 浙江省淡水水产研究所 Enterobacter cloacae specific PCR (polymerase chain reaction) detection primer
CN103305626A (en) * 2013-07-08 2013-09-18 江苏省农业科学院 Primer for detecting escherichia coli pathogenic serum type and detection kit
CN103305627A (en) * 2013-07-16 2013-09-18 中国农业科学院上海兽医研究所 Escherichia coli O1, O2, O18 and O78 serotype detection kit and detection method thereof
CN103540666A (en) * 2013-10-22 2014-01-29 宁波大学 Multiplex PCR (Polymerase Chain Reaction) detection method of enterobacter cloacae in sewage outlet water body

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CN102952881A (en) * 2012-10-25 2013-03-06 浙江省淡水水产研究所 Enterobacter cloacae specific PCR (polymerase chain reaction) detection primer
CN103305626A (en) * 2013-07-08 2013-09-18 江苏省农业科学院 Primer for detecting escherichia coli pathogenic serum type and detection kit
CN103305627A (en) * 2013-07-16 2013-09-18 中国农业科学院上海兽医研究所 Escherichia coli O1, O2, O18 and O78 serotype detection kit and detection method thereof
CN103540666A (en) * 2013-10-22 2014-01-29 宁波大学 Multiplex PCR (Polymerase Chain Reaction) detection method of enterobacter cloacae in sewage outlet water body

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"Structure and genetics of the O-antigen of Enterobacter cloacae C6285 containing di-N-acetyllegionaminic acid";Andrei V. Filatov et al.;《Carbohydrate Research》;20140124;第392卷;第21-24页 *

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