CN112852804A - Universal bacterial target and screening method and application thereof - Google Patents
Universal bacterial target and screening method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of bacteria detection. The invention provides a general target for bacteria, a screening method and application thereof, wherein the sequence of the general target is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7 or SEQ ID NO: shown in fig. 8. The screened 7 bacterial universal targets are used for realizing the direct detection of 15 common pathogenic bacteria in aqueous solution, blood, urine, sputum, orange juice and milk, and the detection limit is as low as 1 CFU/mL. The detection process does not need additional bacterial culture and nucleic acid purification, and the detection result is not influenced by background substances in clinical body fluid samples and common food samples.
Description
Technical Field
The invention relates to the technical field of bacteria detection, in particular to a universal bacterial target and a screening method and application thereof.
Background
The traditional targets for bacterial detection are bacterial bodies and metabolites produced during bacterial culture. The bacteria are detected by using the thalli as a target through a microscope, the detection sensitivity is low, and the detection requirement cannot be met; or the aptamer is taken as a recognition probe, and a sensor is used for detecting the bacterial target, so that the method can improve the detection sensitivity of the bacteria, but the screening of the aptamer of the bacteria is difficult, and only a few kinds of aptamers of the bacteria are screened at present. The metabolite is taken as a target, and is limited by culture conditions and bacterium generation increasing time, so that the positive detection rate is low, and the detection is time-consuming and tedious. In order to meet the demand of rapid detection of bacteria, molecules such as procalcitonin, serum lactic acid, peptidoglycan and nucleic acid are used as bacterial targets. Procalcitonin and serum lactic acid are taken as targets, and the requirements of bacterial detection cannot be met due to insufficient sensitivity and specificity; the method using the peptidoglycan as the target has low detection rate of gram-negative bacteria due to the low content of the peptidoglycan in the cell wall of the gram-negative bacteria. Generally, metabolites, procalcitonin, serum lactic acid and peptidoglycan are taken as targets, and the requirements of quick, accurate and reliable bacterial detection are difficult to meet.
Nucleic acid has the characteristics of stable property and easy editing, and is also used as a bacterial detection target. Nucleic acid targets that have been proposed for bacterial detection are mRNA, 16S rRNA, 23S rRNA, 16-23S rRNA and the like. With the intensive research, mRNA has the characteristics of low abundance existing in bacteria and high mutation speed in the evolution process, and is not suitable for being used as a universal bacterial detection target. Among non-coding protein genes, 16S rRNA provides abundant genetic classification information, and a large amount of 16S rRNA sequence information can be simply obtained from open source databases, so that the gene is an ideal standard target for bacterial detection. The results of 16S rRNA gene sequencing have been used as a basis for bacterial classification. The complete 16S rRNA sequence is used as a target, is only suitable for a gene sequencing method and is not suitable for a hybridization method. The complete 16S rRNA sequence is detected by a hybridization method, and the detection result is inaccurate due to high hybridization mismatch rate. However, the gene sequencing method is difficult to popularize because of high detection cost due to high requirements on operators and instruments.
The 16S rRNA has the structural characteristic of staggered arrangement of a conserved region sequence and a hypervariable region sequence, wherein the conserved region sequence is shared by all bacteria, is highly conserved in the evolution process, and has the potential feasibility of being used as a universal target for detecting the bacteria. The existing targets are 16S rDNA sequence fragments amplified by PCR by using bacterial genome DNA as a template. However, in the annealing step of PCR, there is a problem in accuracy due to base mismatch, resulting in a high false negative and false positive rate of detection. The long sequence fragment is taken as a target to carry out bacterial detection, and the problem of high hybridization mismatch rate exists, so that the detection result is inaccurate. Therefore, a systematic, comprehensive, universal and reliable 16S rRNA conserved fragment universal target database for bacterial detection is established, which is necessary for detection and analysis of microorganisms and can provide universal targets for bacterial detection for researchers.
Disclosure of Invention
The invention aims to provide a universal bacterial target, a screening method and application thereof, and provides a universal bacterial detection target for researchers.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a general target for bacteria, the sequence of which is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7 or SEQ ID NO: shown in fig. 8.
The invention also provides application of the bacterial universal target as a bacterial detection marker.
The invention also provides a screening method of the bacterial universal target, which comprises the following steps:
(1) performing primary screening on the screened object to obtain a 16S rRNA sequence fragment subjected to primary screening;
(2) re-screening the primarily screened 16S rRNA sequence fragments according to an in-strain universality principle, an out-strain specificity principle and a high hybridization efficiency principle to obtain secondarily screened 16S rRNA sequence fragments;
(3) verifying the feasibility of the secondary screened 16S rRNA sequence fragment as a target;
(4) performing a target-based detection of bacteria in the mock sample;
(5) and obtaining a verified 16S rRNA sequence fragment, namely the bacterial universal target.
Preferably, the object to be screened is the full-length 16S rRNA gene sequence of 1414 bacteria described in the clinical microbiology manual.
Preferably, the primary screening is performed by performing multiple sequence homology alignment analysis on the screened objects to find out the base sequences with the sequence length of 13-38 nt and more than 85% of bacteria.
Preferably, the screening criteria of the in-bacterial universality principle are as follows: all strains can be detected by hybridization methods using the sequence fragment as a target.
Preferably, the screening criteria of the out-of-bacteria specificity principle are: targeting the sequence fragment, the probe complementary to the target sequence does not hybridize to a nucleic acid sequence of a non-bacterial species.
Preferably, the screening criteria of the principle of high hybridization efficiency are:
(1) the length is 13-38 nt;
(2) the content of CG is 40-70%;
(3) the Tm value is 55-75 ℃;
(4) when the gene exists in 16S rRNA, no secondary structure exists or the number of ring/stem bases in the secondary structure is less than or equal to 6 nt.
Preferably, the step (3) is performed by PCR-agarose gel electrophoresis.
Preferably, the verification in step (3) includes an in-bacterial universality verification and an out-bacterial specificity study.
The invention provides a general target for bacteria, a screening method and application thereof, wherein the sequence of the general target is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7 or SEQ ID NO: shown in fig. 8. The screened 7 bacterial universal targets are used for realizing the direct detection of 15 common pathogenic bacteria in aqueous solution, blood, urine, sputum, orange juice and milk, and the detection limit is as low as 1 CFU/mL. The detection process does not need additional bacterial culture and nucleic acid purification, and the detection result is not influenced by background substances in clinical body fluid samples and common food samples.
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FIG. 1 is a diagram showing the results of electrophoresis in the general applicability test in example 5;
FIG. 2 is a graph showing the results of electrophoresis in the in vitro specificity study of example 6;
FIG. 3 is the sequence of SEQ ID NO: 1 as a bacterial target, detecting the Escherichia coli in a blood solution sample;
FIG. 4 is the sequence of SEQ ID NO: 2, an electrophoresis result chart for detecting staphylococcus aureus in the blood sample as a bacterial target;
FIG. 5 is the sequence of SEQ ID NO: 3, an electrophoresis result chart for detecting the pseudomonas aeruginosa in the blood sample as a bacterial target;
FIG. 6 is the sequence of SEQ ID NO: 4, an electrophoresis result chart for detecting streptococcus pneumoniae in the sputum sample by taking the streptococcus pneumoniae as a bacterial target;
FIG. 7 is the amino acid sequence of SEQ ID NO: 5, an electrophoresis result chart for detecting the mycobacterium tuberculosis in the urine sample as a bacterial target;
FIG. 8 is the amino acid sequence of SEQ ID NO: 7, an electrophoresis result chart for detecting salmonella enteritidis in the milk sample by taking the bacterial target as the bacterial target;
FIG. 9 is SEQ ID NO: 8 as a bacterial target, and the result chart of electrophoresis for detecting enterohemorrhagic escherichia coli in the orange juice sample.
Detailed Description
The invention provides a general target for bacteria, the sequence of which is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7 or SEQ ID NO: shown in fig. 8.
The invention also provides application of the bacterial universal target as a bacterial detection marker.
The invention also provides a screening method of the bacterial universal target, which comprises the following steps:
(1) performing primary screening on the screened object to obtain a 16S rRNA sequence fragment subjected to primary screening;
(2) re-screening the primarily screened 16S rRNA sequence fragments according to an in-strain universality principle, an out-strain specificity principle and a high hybridization efficiency principle to obtain secondarily screened 16S rRNA sequence fragments;
(3) verifying the feasibility of the secondary screened 16S rRNA sequence fragment as a target;
(4) performing a target-based detection of bacteria in the mock sample;
(5) and obtaining a verified 16S rRNA sequence fragment, namely the bacterial universal target.
In the present invention, the object to be screened is preferably the full-length 16S rRNA gene sequence of 1414 bacteria described in the clinical microorganism manual.
In the invention, during the primary screening, multiple sequence homology comparison analysis is carried out on the screened objects, and the base sequences with the sequence length of 13-38 nt and more than 85% of bacteria are found.
In the present invention, the screening criteria of the universal principle are preferably: all strains can be detected by hybridization methods using the sequence fragment as a target.
In the present invention, the screening process of the universal principle is preferably as follows: introducing the screened object into a 16S ribosomal RNA sequences (16S) database of NCBI, setting the comparison parameter as Highly similar sequences (megablast), displaying the result as 5000, setting other parameters as system defaults, outputting the comparison result, and if the output result is less than 5000, not meeting the screening requirement; if the output result is 5000, further setting the parameters Identity to be 85-100% and cover to be 100%, performing comparison, and taking the sequence with the output result of 5000 after comparison.
In the present invention, the screening criteria of the out-of-bacteria specificity principle are preferably: targeting the sequence fragment, the probe complementary to the target sequence does not hybridize to a nucleic acid sequence of a non-bacterial species.
In the present invention, the screening process of the out-of-bacteria specificity principle is preferably as follows: introducing a screening object into a fungus database and a Human Gene database of NCBI, setting the similarity as highlysimilar sequences (megablast), fungus 18S ribosomal RNA sequences (18S) and a Human Ref Seq Gene database, comparing the matching degree of the preliminarily screened sequence fragments with the sequences in the fungus and Human genome database, setting the comparison parameters as highlysimilar sequences (megablast), displaying the result as 100, setting other parameters as system defaults, outputting the comparison result, further setting the parameters Identity as 85-100% and the cover as 100%, comparing, and taking the sequence of which the output result after comparison is 0.
In the present invention, the screening criteria of the principle of high hybridization efficiency are:
(1) the length is 13-38 nt;
(2) the content of CG is 40-70%;
(3) the Tm value is 55-75 ℃;
(4) when the gene exists in 16S rRNA, no secondary structure exists or the number of ring/stem bases in the secondary structure is less than or equal to 6 nt.
In the present invention, it is preferable that the verification in the step (3) is performed by PCR-agarose gel electrophoresis.
In the present invention, the verification in step (3) includes an in-bacterial universality verification and an out-bacterial specificity study.
In the invention, the process of the in-bacterial universality verification is preferably as follows: taking the universal primer 27F of the bacteria accepted in the industry and the reverse complementary sequence of the sequence fragment of the screened 16S rRNA as a pair of primers required by PCR, and taking 15 common pathogenic strains (the concentration of the bacteria is (1.3 +/-0.5) × 10)5CFU/ml) 16S rRNA is used as a template, PCR amplification is carried out, an agarose gel electrophoresis method is used for detecting an amplification product, and if an electrophoresis band appears, the sequence to be detected is successfully hybridized with the primer, so that the target for detecting bacteria can be obtained.
In the present invention, the process of the extrabacterial specificity study is preferably: uses commonly accepted fungus primer NS1 and reverse complementary sequence of screened 16S rRNA sequence fragment as a pair of primers required by PCR, and uses Candida albicans, Candida glabrata, Nocardia asteroides, and Saccharomyces cerevisiae (bacterial concentration is (1.1 +/-0.5) × 10)5CFU/ml) 18S RNA is used as a template for PCR amplification; and (3) detecting the amplification product by using an agarose gel electrophoresis method, and if no electrophoresis band appears, indicating that the sequence to be detected does not exist in the fungal genome and can be used as a bacterial detection target.
In the present invention, the process of performing the detection of bacteria in the target-based mock sample is preferably: after the target sequence is determined, a reverse complementary nucleic acid sequence is synthesized, and the feasibility of detecting bacteria in various environmental samples by taking the sequence as a target is verified through a PCR-agarose gel electrophoresis technology.
In the present invention, the sample environment tested in the process of performing the detection of bacteria in the target-based mock sample is preferably clinical body fluids and food drinks.
In the present invention, the clinical body fluid is preferably blood, urine, saliva, a throat swab.
In the invention, the food and drink is preferably milk or orange juice.
In the invention, the tested target bacteria are preferably blood culture instrument industry standard strains, fastidious bacteria and food-borne pathogenic bacteria.
In the present invention, the hemoculture apparatus industry standard strain is preferably Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Acinetobacter baumannii, Salmonella enteritidis, Staphylococcus epidermidis, Enterobacter aerogenes, Salmonella paratyphi A, enterococcus faecium, enterococcus faecalis, Enterobacter cloacae, Klebsiella pneumoniae by plantation, Serratia marcescens, stenotrophomonas maltophilia.
In the present invention, the fastidious bacteria are preferably streptococcus pneumoniae and mycobacterium tuberculosis.
In the present invention, the food-borne pathogenic bacteria are preferably Salmonella enteritidis and enterohemorrhagic Escherichia coli.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 Primary Screen Using NCBI GenBank database
16S rRNA gene full-length sequences of 1414 clinical bacteria are searched and downloaded through an NCBI GenBank database, and the downloaded sequences are introduced into sequence alignment software DNAMAN for multi-sequence homology alignment analysis. Finding out 31 base sequences with the sequence length of 13-38 nt and more than 85% of bacteria, wherein the sequences are shown as SEQ ID NO: 1 to SEQ ID NO: shown at 31.
Example 2 Primary screening of 16S rRNA sequence fragments according to the principles of in-bacterial universality
The 31 sequences obtained in the primary screening were introduced into the 16S ribosomal RNA sequences (16S) database of NCBI, the alignment parameters were set to high similarity sequences (megablast) showing a result of 5000 (upper output limit), the other parameters were default of the system, and the selected sequences were aligned to match the 16S rRNA of the bacteria in the database. And further screening out sequence fragments with matching degrees of 85-100% for Identity and 100% for cover, and outputting the sequence fragments with the result of 5000, wherein the results are shown in table 1.
TABLE 1
Example 3 Primary screening of 16S rRNA sequence fragments according to the Ex-bacterial specificity principle
The 18 sequence fragments selected in example 2 were introduced into the fungus database and human gene database of NCBI, similarity was set to Highly similar sequences (megablast), other parameters were set as system defaults, sequence fragments satisfying Identity 85-100%, Cover 100% and 0 were selected, and the results are shown in table 2.
TABLE 2
Example 4 Primary screening of 16S rRNA sequence fragments according to high hybridization efficiency
The sequences obtained in Table 2 in example 3 were introduced into Array design software, and sequence fragments having a CG content of 40% -70%, a Tm value of 55-75 ℃ and having no secondary structure in 16S rRNA or having a number of loop/stem bases of 6nt or less in the secondary structure were selected, and the results are shown in Table 3.
TABLE 3
Example 5 in-bacterial universality validation
Is prepared from Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Acinetobacter baumannii, and intestinal bacteriaSalmonella enteritidis, staphylococcus epidermidis, enterobacter aerogenes, salmonella paratyphi A, enterococcus faecium, enterococcus faecalis, enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens and stenotrophomonas maltophilia are taken as bacteria representatives, and PCR electrophoresis experiments are utilized to verify the hybridizability of the sequences shown in the table 3 contained in the bacteria in a water sample. Taking 200 μ L of 103CFU/mL of an aqueous sample of the above bacteria was heated for 80s with a microwave oven medium fire. Adding 1 mu L of the treated bacterial liquid into 1 mu L of 10 mu M reverse complementary sequence of the target to be detected, 1 mu L of 10 mu M standard universal primer 27F, 10 mu LPCR required mixed solution and 12 mu LddH2O in a PCR tube, and setting PCR parameters as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. As shown in FIG. 1, lanes 1 to 11 represent the PCR results for the target sequences 1 to 11 in Table 3, respectively, the leftmost channel represents the DNA marker, and the rightmost channel represents the PCR results for the solution containing 27F + lytic bacteria. Sequence SEQ ID NO: 1 to SEQ ID NO: 5. SEQ ID NO: 7 and SEQ ID NO: 8 meets the universality requirement, and the sequence SEQ ID NO: 6. SEQ ID NO: 9. SEQ ID NO: 10 and SEQ ID NO: and 11, the universality requirement is not met.
The corresponding bacteria in fig. 1 are: (a) escherichia coli, (b) staphylococcus aureus, (c) pseudomonas aeruginosa, (d) streptococcus pneumoniae, (e) acinetobacter baumannii, (f) salmonella enteritidis, (g) staphylococcus epidermidis, (h) enterobacter aerogenes, (i) salmonella paratyphi, (j) enterococcus faecium, (k) enterobacter cloacae, (l) klebsiella pneumoniae, (m) serratia marcescens, (n) stenotrophomonas maltophilia, and (o) enterococcus faecalis.
Example 6 Ex-bacterial specificity Studies
Candida albicans, Candida glabrata, Cryptococcus neoformans, Nocardia asteroides and Saccharomyces cerevisiae are used as fungi representatives, and the specificity outside the bacteria of the 16S rRNA sequence segment which meets the universality inside the bacteria and is screened out in the example 5 is verified by utilizing a PCR electrophoresis experiment. Taking 200 μ L of 103CFU/mL of the above fungal aqueous solution sample was heated for 80s with a microwave oven medium fire. Adding 1 μ L of the treated bacterial liquid into the mixtureL10. mu.M of the reverse complement of the target to be detected, 1. mu.L 10uM of the fungal Standard Universal primer NS1, 10. mu.L of the mixture required for PCR, 12. mu.L of ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. PCR products were obtained, and the results of PCR were verified by agarose gel electrophoresis, as shown in FIG. 2. Lanes 1-7 represent the sequences represented by SEQ ID NOs: 1 to SEQ ID NO: 5. SEQ ID NO: 7 and SEQ ID NO: 8 as the target of the PCR results, the leftmost channel represents the DNA marker (100-. The results show that the sequence SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7 and SEQ ID NO: 8 meet the specificity requirement.
The corresponding bacteria in fig. 2 are: (a) candida albicans, (b) Candida glabrata, (c) Cryptococcus neoformans, (d) Planet nocardia minor, (e) Saccharomyces cerevisiae.
Example 7 SEQ ID NO: 1 detection of Escherichia coli in blood solution sample as bacterial target
100 μ L of blood was added to 900 μ L of a solution containing 1X 103And preparing a bacterium-containing simulated blood sample from the bacterial liquid of the CFU/mL escherichia coli. Centrifuging the blood sample at 12000r/min for 3min, collecting precipitate, and heating with microwave oven at middle fire for 80 s. 1 mu L of the treated bacterial liquid is taken, and 1 mu L of 10 mu M SEQ ID NO: 1, 1. mu.L of 10. mu.M standard universal primer 27F, 10. mu.L of the PCR mix, 12. mu.L of ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. Results as shown in FIG. 3, lane 0 represents the PCR results for 27F + lysed bacteria, referred to as blank; lane m represents the DNA marker (100-10000bp), lane 1 represents the DNA marker represented by SEQ ID NO: 1 as the result of PCR for the target. In FIG. 3, lane 0 shows no bands, indicating that background interference does not affect the detection results; lane 1 shows a band indicating the presence of SEQ ID NO: 1 is a bacterial target, and realizes the detection of Escherichia coli in blood samples.
Example 8 SEQ ID NO: 2 detecting staphylococcus aureus in blood sample as bacterial target
100 μ L of blood was added to 900 μ L of a solution containing 1X 103And preparing a bacterium-containing simulated blood sample from the bacterial liquid of the CFU/mL staphylococcus aureus. Centrifuging the blood sample at 12000r/min for 3min, collecting precipitate, and heating with microwave oven at middle fire for 80 s. 1 mu L of the treated bacterial liquid is taken, and 1 mu L of 10 mu M SEQ ID NO: 2, 1. mu.L of 10uM standard universal primer 27F, 10. mu.L of the desired cocktail of LPCR, 12. mu.L ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. Results as shown in FIG. 4, lane 0 represents the PCR results for 27F + lysed bacteria, referred to as blank; lane m represents the DNA marker (100-10000bp), lane 2 represents the DNA marker represented by SEQ ID NO: 2 as the result of PCR for the target. In FIG. 4, no band appears in lane 0, indicating that background interference does not affect the detection result; lanes 2 all show bands indicating the presence of SEQ ID NO: 2 is a bacterial target, and realizes the detection of staphylococcus aureus in blood samples.
Example 9 SEQ ID NO: 3 as a bacterial target to detect the pseudomonas aeruginosa in the blood sample
100 μ L of blood was added to 900 μ L of a solution containing 1X 103Preparing a bacterium-containing simulated blood sample from the bacterial solution of the CFU/mL pseudomonas aeruginosa. Centrifuging the blood sample at 12000r/min for 3min, collecting precipitate, and heating with microwave oven at middle fire for 80 s. 1 mu L of the treated bacterial liquid is taken, and 1 mu L of 10uM SEQ ID NO: 3, 1. mu.L of 10uM standard universal primer 27F, 10. mu.L of the desired cocktail of LPCR, 12. mu.L ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. Results as shown in FIG. 5, lane 0 represents the PCR results for 27F + lysed bacteria, referred to as blank; lane m represents the DNA marker (100-10000bp), lane 3 represents the DNA marker represented by SEQ ID NO: 3 as the result of PCR for the target. In FIG. 5, the swimming strokeNo band appears in the channel 0, which indicates that the background interference does not influence the detection result; lane 3 shows a band indicating the presence of SEQ ID NO: 3 is a bacterial target, and realizes the detection of the pseudomonas aeruginosa in the blood sample.
Example 10 SEQ ID NO: 4 detecting streptococcus pneumoniae in sputum sample as bacterial target
The sputum is collected by a natural expectoration method. Adding 100 μ L of sputum to 900 μ L of sputum containing 1 × 103And preparing a bacteria-containing simulated sputum sample from the bacterial solution of the CFU/mL streptococcus pneumoniae. Centrifuging the sputum sample at 12000r/min for 3min, collecting precipitate, and heating with microwave oven at middle fire for 80 s. 1 mu L of the treated bacterial liquid is taken, and 1 mu L of 10 mu M SEQ ID NO: 4, 1. mu.L of 10uM standard universal primer 27F, 10. mu.L of the desired cocktail of LPCR, 12. mu.L of ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. Results as shown in FIG. 6, lane 0 represents the PCR results for 27F + lysed bacteria, referred to as blank; lane m represents the DNA marker (100-10000bp), lane 4 represents the DNA marker represented by SEQ ID NO: 4 as the result of PCR for the target. In FIG. 6, no band appears in lane 0, indicating that background interference does not affect the detection result; lane 4 shows a band indicating the presence of SEQ ID NO: 4 as a target, realizes the detection of the streptococcus pneumoniae in the sputum sample.
Example 11 SEQ ID NO: 5 as a bacterial target to detect the mycobacterium tuberculosis in the urine sample
Urine was collected by sterile catheterization. Adding 100 μ L urine to 900 μ L urine containing 1 × 103And preparing a bacteria-containing simulated urine sample from the bacterial liquid of the CFU/mL mycobacterium tuberculosis. Centrifuging the urine sample at 12000r/min for 3min, collecting precipitate, and heating with microwave oven at middle fire for 80 s. 1 mu L of the treated bacterial liquid is taken, and 1 mu L of 10 mu M SEQ ID NO: 5, 1. mu.L of 10. mu.M standard universal primer 27F, 10. mu.L of the PCR mix, 12. mu.L of ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; extension temperature of 72 DEG CTime 90s, cycle 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. Results as shown in FIG. 7, lane 0 represents the PCR results for 27F + lysed bacteria, referred to as blank; lane m represents the DNA marker (100-10000bp), lane 5 represents the DNA marker represented by SEQ ID NO: 5 PCR results for the target. In FIG. 7, no band appears in lane 0, indicating that background interference does not affect the detection result; lane 5 shows a band indicating the presence of SEQ ID NO: 5 is used as a target, so that the detection of the mycobacterium tuberculosis in the urine sample is realized.
Example 12 SEQ ID NO: 7 detecting salmonella enteritidis in milk sample as bacterial target
Adding 100 μ L milk into 900 μ L milk containing 1 × 103And preparing a bacteria-containing simulated milk sample from the bacterial solution of the CFU/mL salmonella enteritidis. Centrifuging milk sample at 12000r/min for 3min, collecting precipitate, and heating with microwave oven at middle fire for 80 s. 1 mu L of the treated bacterial liquid is taken, and 1 mu L of 10 mu M SEQ ID NO: 7, 1. mu.L of 10. mu.M standard universal primer 27F, 10. mu.L of the PCR mix, 12. mu.L of ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. Results as shown in FIG. 8, lane 0 represents the PCR results for 27F + lysed bacteria, referred to as blank; lane m represents the DNA marker (100-10000bp), lane 6 represents the DNA marker represented by SEQ ID NO: 7 PCR results for the target. In FIG. 8, no band appears in lane 0, indicating that background interference does not affect the detection result; lane 6 shows a band indicating the presence of SEQ ID NO: 7 is used as a target, so that the detection of salmonella enteritidis in the milk sample is realized.
Example 13 SEQ ID NO: 8 as a bacterial target to detect enterohemorrhagic escherichia coli in the orange juice sample
Adding 100 μ L orange juice into 900 μ L orange juice containing 1 × 103And (4) preparing a bacteria-containing simulated orange juice sample from the bacterial solution of the CFU/mL enterohemorrhagic escherichia coli. Centrifuging the orange juice sample at 12000r/min for 3min, collecting precipitate, and heating with microwave oven at middle fire for 80 s. 1 mu L of the treated bacterial liquid is taken, and 1 mu L of 10 mu M SEQ ID NO:8, 1. mu.L of 10. mu.M standard universal primer 27F, 10. mu.L of the PCR mix, 12. mu.L of ddH2Setting PCR parameters in a PCR tube as follows: the denaturation temperature is 94 ℃ and the time is 5 min; the renaturation temperature is 55 ℃, and the time is 30 s; the extension temperature is 72 ℃, the time is 90s, and the cycle is 30 times. Obtaining PCR products, and verifying the PCR results by agarose gel electrophoresis. Results as shown in FIG. 9, lane 0 represents the PCR results for 27F + lysed bacteria, referred to as blank; lane m represents the DNA marker (100-10000bp), lane 7 represents the DNA marker represented by SEQ ID NO: 8 PCR results for the target. In FIG. 9, lane 0 shows no bands, indicating that background interference does not affect the detection results; lane 7 shows a band indicating the presence of SEQ ID NO: 8 is used as a target, so that the detection of enterohemorrhagic escherichia coli in the orange juice sample is realized.
The above embodiments show that the present invention provides a bacterial universal target, a screening method and an application thereof, and the sequence thereof is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7 or SEQ ID NO: shown in fig. 8. The screened 7 bacterial universal targets are used for realizing the direct detection of 15 common pathogenic bacteria in aqueous solution, blood, urine, sputum, orange juice and milk, and the detection limit is as low as 1 CFU/mL. The detection process does not need additional bacterial culture and nucleic acid purification, and the detection result is not influenced by background substances in clinical body fluid samples and common food samples.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)
1. A bacterial universal target having a sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7 or SEQ ID NO: shown in fig. 8.
2. Use of the universal bacterial target of claim 1 as a marker for bacterial detection.
3. The method for screening a universal target for a bacterium according to claim 1, comprising the steps of:
(1) performing primary screening on the screened object to obtain a 16S rRNA sequence fragment subjected to primary screening;
(2) re-screening the primarily screened 16S rRNA sequence fragments according to an in-strain universality principle, an out-strain specificity principle and a high hybridization efficiency principle to obtain secondarily screened 16S rRNA sequence fragments;
(3) verifying the feasibility of the secondary screened 16S rRNA sequence fragment as a target;
(4) performing a target-based detection of bacteria in the mock sample;
(5) and obtaining a verified 16S rRNA sequence fragment, namely the bacterial universal target.
4. The method for screening a universal target for a bacterium according to claim 3, wherein the object to be screened is the full-length 16S rRNA gene sequence of 1414 bacteria described in a clinical microbiology manual.
5. The method of claim 4, wherein the primary screening is performed by performing multiple sequence homology alignment analysis on the screened objects to find out the base sequences with a sequence length of 13-38 nt and more than 85% of the bacteria.
6. The method for screening universal bacterial targets of claim 5, wherein the screening criteria of the in-bacterial universality principle are as follows: all strains can be detected by hybridization methods using the sequence fragment as a target.
7. The method for screening a universal target of bacteria according to claim 6, wherein the screening criteria of the out-of-bacteria specificity principle are: targeting the sequence fragment, the probe complementary to the target sequence does not hybridize to a nucleic acid sequence of a non-bacterial species.
8. The method for screening a universal target of bacteria according to claim 7, wherein the screening criteria of the principle of high hybridization efficiency are:
(1) the length is 13-38 nt;
(2) the content of CG is 40-70%;
(3) the Tm value is 55-75 ℃;
(4) when the gene exists in 16S rRNA, no secondary structure exists or the number of ring/stem bases in the secondary structure is less than or equal to 6 nt.
9. The method for screening a universal target of bacteria according to claim 8, wherein the step (3) is performed by PCR-agarose gel electrophoresis.
10. The method for screening a universal target of bacteria according to any one of claims 3 to 9, wherein the verification in step (3) comprises an in-bacterial universal verification and an out-bacterial specificity study.
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