CN1069925C - Salmonella rDNA spacer nucleic acid molecular probe and its using method - Google Patents

Abstract

The present invention relates to an rDNA spacer ribonucleic acid molecular probe for detecting salmonella, which belongs to molecular biology. An rDNA central African RNA encoding region of salmonella is used as detection target genes, and five groups of 9 kinds of salmonella are used as development materials. The rDNA spacer ribonucleic acid molecular probe is synthesized by a conventional DNA synthesis method, the structure comprises 23 ribonucleotides, and the nucleotide sequence is 5'-CGAAGCATACATCAGTATGTTAG-3'. The probe can be used for detecting salmonella in various samples of foods, medicines, environment, etc. by a ribonucleic acid molecular hybridization method, a conventional PCR method and a reference primer PCR method, and furthermore, the probe has the advantages of simple and convenient operation, high speed, precision, sensitivity, no poison, etc.

Description

Salmonella rDNA spacer nucleic acid molecular probe and application method thereof

The invention relates to molecular biology, specifically a biological probe for testing salmonella, which can be directly applied to food hygiene inspection, environmental testing, customs quarantine and salmonella testing in clinical medicine.

There are many types of Salmonella, and more than 2,000 species have been found to be common pathogenic bacteria. Salmonella inspection is a mandatory inspection item for food hygiene and environmental quarantine. At present, the Salmonella inspection method promulgated by the Ministry of Health of my country is mainly based on the morphology, physiological and biochemical characteristics and serum reactions of Salmonella. The process is complicated, the operation is troublesome, and the period It is long and prone to cross-reaction with close bacteria to affect the accuracy. Even also will use highly toxic reagent (potassium hydride) in existing salmonella detection method, has hazard to the health of staff.

So far, China has not published any research and patents on Salmonella nucleic acid molecular probes, but the developed countries in the world have invested a lot of money and manpower in the development of new detection methods and nucleic acid molecular probes for Salmonella. In rDNA nucleic acid molecular probes, there are two different types of probes, rRNA coding region probes and non-RNA coding region probes. A kind of Salmonella rDNA probe is disclosed in U.S. Patent VS5147778, and it is the target gene of detection with the sequence of rRNA coding region in rRNA or rDNA, although it has established a kind of rapid, nontoxic Salmonella detection method, sensitivity is still not enough high. It is very difficult to find detection targets with higher specificity, and it is even more difficult to develop probes. Researchers such as the United States and Japan are developing in this direction. But at present, there are no relevant research reports at home and abroad.

The object of the present invention is to overcome the weak point that background technology exists, apply the gene detection and identification technology based on nucleic acid molecular probe to the inspection of Salmonella, develop a kind of specificity higher nucleic acid molecular probe and its application method. Therefore, a new method for the detection of Salmonella, which is fast, accurate, non-toxic and highly sensitive, was established.

A Salmonella nucleic acid molecular probe is characterized in that the non-RNA coding region in the Salmonella rDNA is used as the detection target gene, and the Salmonella rDNA spacer nucleic acid molecular probe is designed. Its structure is composed of 23 nucleotides, and its nucleotide The sequence is as follows: 5'CGAAGCATACATCAGTATGTTAG 3'.

rRNA and its gene rDNA are a good target gene for the development of Salmonella nucleic acid molecular probes. There are rRNA coding regions and non-RNA coding regions (i.e. spacers) in rDNA. The rRNA coding region sequences in rRNA sequences and rDNA can be It is used to develop Salmonella rRNA probes and rDNA probes, and the non-RNA coding region in rDNA can be used to develop Salmonella rDNA spacer nucleic acid molecular probes.

Bacterial rDNA spacer is a hypervariable region, and the rDNA spacer structure of different bacterial groups is very different, and this difference can also reflect the distance of kinship between different bacteria (that is, the farther the kinship, the greater the difference, and vice versa ). In the rDNA spacer region, there is a completely different nucleotide sequence between Salmonella and other non-Salmonella, that is, the characteristic nucleotide sequence of Salmonella. The Salmonella rDNA spacer nucleic acid molecular probe established according to this characteristic sequence has a very high specificity for Salmonella. oneness. The probe can specifically react with Salmonella DNA, but does not cross-react with other non-Salmonella DNA, so it can be used for the identification of Salmonella and the detection of Salmonella in samples.

To establish the Salmonella rDNA spacer nucleic acid molecular probe, the first step is to carry out molecular cloning and DNA sequence determination of the Salmonella rDNA spacer, and to investigate the different species of Salmonella. Finally, according to the results of DNA sequence determination and analysis, design and Synthesize the Salmonella rDNA spacer nucleic acid molecular probe, which can be tested by molecular hybridization or PCR. If there is Salmonella, a positive sign will appear, and if there is no Salmonella, a negative sign will appear.

According to the nucleotide sequence of the Salmonella rDNA spacer determined by the inventor, and compared with other non-Salmonella rDNA sequence data published. The present invention selects five groups of 9 species of Salmonella to develop the Salmonella rDNA spacer nucleotide molecular probe. These five groups of 9 species of Salmonella all come from the pathogenic bacteria room of the Guangzhou Epidemic Prevention Station. Their Chinese and Latin names are as follows:

Group A (A):

Salmonella paratyphi A

Group B (B):

Salmonella paratyphi B (S.paratyphi B)

Salmonella typhimurium (S.typhi murium)

Group C (C1):

Salmonella paratyphi C (S.paratyphi C)

Salmonella Tennessee (S.tennessee)

Group D (D):

Salmonella typhi (S.typhi)

Salmonella Enteritidis (S. enteritidis)

E (E1) group:

Duck Salmonella (S.anatum)

Salmonella weltevreden (S.weltevreden)

The present invention is composed of 23 nucleotides, and its nucleotide sequence is 5'CGAAGCATACATCAGTATGTTAG 3' Salmonella rDNA spacer nucleic acid molecular probe can be mass-produced by any conventional DNA synthesis method, for example : It can be synthesized using a commercialized DNA automatic synthesizer.

The Salmonella rDNA spacer nucleic acid molecular probe developed by the present invention can be tested for Salmonella in various samples such as food, medicine, environment, etc. by nucleic acid molecular hybridization method or conventional PCR method, and the probe is only specific to Salmonella DNA. Reaction, but no cross-reaction with non-Salmonella DNA. In addition, a unique reference primer PCR method of the present invention was established.

The above application methods are introduced respectively as follows:

(1) Nucleic acid molecular hybridization method

The Salmonella rDNA spacer nucleic acid molecular probe can use various nucleic acid molecular hybridization methods (such as dot hybridization) to detect Salmonella in a sample or identify a known bacterium.

The process of hybridization of nucleic acid molecules (including labeling of probes and pretreatment of samples to be tested) is carried out according to conventional molecular biology methods.

The nucleic acid hybridization method can be used for direct inspection of samples in food, medicine, and the environment, or the Salmonella DNA in the sample can be amplified first, and then inspected. Non-Salmonella in the sample does not interfere with the detection of Salmonella rDNA spacer nucleic acid molecular probes. Specific detection of Salmonella.

(two), conventional PCR method

The Salmonella rDNA spacer nucleic acid molecular probe can be used as a PCR primer to pair with another PCR primer, such as the PCR method of the bacterial rDNA universal primer, to quickly and accurately detect a small amount of Salmonella DNA in the sample. It does not interfere with the specific detection of Salmonella rDNA spacer nucleic acid molecular probes.

The process of carrying out PCR detection of Salmonella by using the Salmonella rDNA spacer nucleic acid molecular probe can be carried out according to the conventional PCR operation procedures.

(3), reference primer PCR method

Using more than one pair of primers with different specificities to carry out PCR reaction on the same sample in the same test tube, the target sequences amplified by different primers are different, and the molecular weights of the PCR products are also significantly different according to the design. According to the type of the final PCR product in a test tube, it can be judged whether the sample contains Salmonella, or other bacteria and whether it is sterile. Because the PCR products of different primer pairs can be referred to each other, it is called the reference primer PCR method. The results of PCR method can be checked by agarose gel electrophoresis.

The reference primer PCR method is used for the detection of Salmonella, and the PCR primer selection settings are as follows:

① Set a bacterial universal PCR primer in the 3' terminal conservative region of the bacterial rDNA 16S rRNA coding region, and set another primer with Salmonella specificity in the spacer in the rDNA, that is, between the Salmonella rDNA spacer nucleic acid molecular probe and the primer The distance is 207 nucleotides.

② Set another pair of bacterial rDNA universal primers in the 5' end conserved region of the bacterial rDNA 16S rRNA coding region, and the distance between them is 950 nucleotides.

Fig. 1 is the electrophoresis result of five kinds of Salmonella such as typhoid murine, salmonella cold etc. that adopt conventional PCR method to check with Salmonella rDNA spacer nucleic acid molecule probe for embodiment 1.

Fig. 2 is the electrophoresis result of five kinds of Salmonella such as Salmonella paratyphi A using reference primer PCR method to check with Salmonella rDNA spacer nucleic acid molecular probe in Example 2.

Invention effect:

The invention of the Salmonella rDNA spacer nucleic acid molecular probe and its application method represents a new method for genetic identification and detection of Salmonella, which is based on a completely different principle from the current conventional method. The new method has the advantages of simplicity, rapidity, accuracy, sensitivity and non-toxicity. It can be used to strengthen or replace the routine Salmonella inspection method promulgated by the Ministry of Health of my country.

Compared with the current Salmonella detection method, the new method has the following advantages:

1. Simple and fast: routine inspection is cumbersome, and the experiment period is long (sometimes as long as a week), while the new method only takes a few hours (if the amplification of bacteria is required, the amplification time is excluded).

2. Accurate: The Salmonella rDNA spacer nucleic acid molecular probe only hybridizes with Salmonella rDNA, but does not cross-react with non-Salmonella DNA.

3. High sensitivity: the Salmonella rDNA spacer nucleic acid molecular probe can be used in combination with the PCR method, and the theoretical detection value of Salmonella can reach one bacterium.

4. Do not use toxic reagents.

Example 1

1. Preparation of nucleic acid molecular probes and PCR primers

①According to the nucleotide sequence of 5′CGAAGCATACATCAGTATGTTAG 3, an automatic DNA synthesizer was used to synthesize the Salmonella rDNA spacer nucleic acid molecular probe using a conventional DNA synthesis method.

②Salmonella rDNA spacer nucleic acid molecular probe (Salmonella specific) and other three bacterial rDNA PCR universal primers P ₁₄₈₆ (20 nucleotides long), P ₁₆₊ (22 nucleotides long), P _16- (22 nucleotides in length), all synthesized by an automatic DNA synthesizer, dissolved in TE (Tris-Hcl Tomm, EDTA 1mmPH7.6) solution at a concentration of 0.5 μg/μl.

2. Salmonella rDNA spacer nucleic acid molecular probe inspection process of Salmonella (conventional PCR method)

①Salmonella sample pretreatment:

Take 1 ml of culture solution containing Salmonella ( ¹⁰⁵ bacteria) and put it into a 1.5 ml small finger tube, centrifuge (5000 rpm) for 2 minutes, pour off the supernatant, and add an appropriate amount of 0.2M sodium hydroxide (to dissolve the bacteria) in the small tube Intensity), immediately add 500 microliters of TE solution (PH7.0) and then dilute to 1000 bacteria/microliter according to the number of bacteria.

② 100 microliters of No. 1 PCR mixture:

Salmonella rDNA spacer nucleic acid molecular probe 0.5 μg

P ₁₄₈₆ Nucleic Acid Molecular Probe 0.5 μg

10X Tap DNA Polymerase Buffer 10 microliters

2mmdNTP solution 10 microliters

Tap DNA Polymerase 2.5 units

Dilute to 100 microliters with sterile water

③ 100 microliters of the ingredients in the No. Ⅱ PCR mixture

Bacterial rDNA PCR universal primers P _16S+ and P _16S- were used to replace Salmonella rDNA spacer nucleic acid molecular probe and P ₁₄₈₆ respectively, and other components were the same as No. Ⅰ PCR mixture.

④PCR operation

Take nine 0.5 ml small finger tubes, add 20 microliters of No. 1 PCR mixture to tubes 1 to 8, and then add 5 kinds of Salmonella samples including Salmonella typhimurium and Escherichia coli (control). Add 20 microliters of No. 2 PCR mixture into tube 9, and then add Escherichia coli sample (control). Add 50 microliters of mineral oil to each tube, put it on the PCR instrument, and carry out the PCR reaction according to the following procedure:

94°C for 5 minutes, 94°C, 50°C, and 72°C for 1 minute each, and cycled continuously 30 times, then 72°C for 5 minutes.

After the reaction, 5 microliters of loading solution (30% Ficol 0.25% bromophenol blue) was added to each tube.

Take 4 microliters and spot on 2% agarose gel, and the electrophoresis result is shown in Figure 1.

Figure 1 shows that:

1, 2, 12:1Kb molecular weight gradient

3. Salmonella typhimurium (equivalent to 40 bacteria)

4. Salmonella Tennessee (equivalent to 40 bacteria)

5. Sterile water

6. Salmonella enteritidis (equivalent to 40 bacteria)

7. Duck Salmonella (equivalent to 40 bacteria)

8. Salmonella Wertfrieden (equivalent to 40 bacteria)

9. Escherichia coli (equivalent to 400 bacteria)

10. Escherichia coli (equivalent to 40 bacteria)

11. Escherichia coli (equivalent to 40 bacteria)

Using the PCR primer pair composed of the Salmonella rDNA spacer nucleic acid molecular probe and P ₁₄₈₆ , five different Salmonella (No. 3, 4, 6, 7, and 8) such as typhoid fever can be accurately detected (with 207 nucleotides) positive flag) and no cross-reaction with Escherichia coli (No. 9, No. 10). The PCR primer pair composed of P _16S+ and P _16S- was used to test the same Escherichia coli DNA sample (No. 11) as the No. 10 sample. As a control, a positive mark of 950 nucleotides appeared, indicating the presence of bacterial DNA.

Example 2

1, the preparation of nucleic acid molecular probe and PCR primer is the same as in Example 1

2. Use the Salmonella rDNA spacer nucleic acid molecular probe to test the Salmonella operation (refer to the primer PCR method)

1. Salmonella sample pretreatment is the same as in Example 1

② 100 microliters No. Ⅲ PCR mixture composition:

Salmonella rDNA spacer nucleic acid molecular probe 0.5 μg

P ₁₄₈₆ Nucleic Acid Molecular Probe 0.5 μg

P ₁₆₊ Nucleic Acid Molecular Probe 0.5 μg

P _16- nucleic acid molecular probe 0.5 μg

10X Tap DNA Polymerase Buffer 10 microliters

2mmdNTP solution 10 microliters

Tap DNA Polymerase 2.5 units

Make up to 100 microliters with water

③PCR operation

Take nine 0.5 ml small finger tubes, add 20 microliters of the above PCR mixture to each, then add various Salmonella samples such as Salmonella paratyphi A and E. coli samples or sterile water, and cover each tube with 50 microliters of mineral oil , operating procedure with embodiment 1. The results of electrophoresis are shown in Figure 2.

Figure 2 shows that:

1. Salmonella typhi (equivalent to 40 bacteria)

2. Salmonella paratyphi C (equivalent to 40 bacteria)

3. Salmonella typhi (equivalent to 100 bacteria)

4. Salmonella paratyphi C (equivalent to 100 bacteria)

5. Salmonella paratyphi B (equivalent to 100 bacteria)

6. Salmonella paratyphi A (equivalent to 100 bacteria)

7. Salmonella typhimurium (equivalent to 40 bacteria)

8. Escherichia coli (equivalent to 40 bacteria)

11. 1Kb molecular weight gradient

Using two pairs of primers, that is, the reference primer PCR method of Salmonella rDNA spacer nucleic acid molecular probe/P ₁₄₈₆ and P ₁₆₊ /P _16- , ① There should be at least one Salmonella positive marker of 207 nucleotides in samples containing Salmonella, and in addition , another bacterial positive marker of 950 nucleotides can also appear, but in some samples, the bacterial positive marker of 950 nucleotides is weaker. ② In the sample (No. 8) that does not contain Salmonella but contains Enterobacter, only 950 nucleotides of bacterial positive markers appear. ③ In the sterile sample, there is no DNA band (No. 9).

Claims (2)

1. Salmonella nucleic acid molecular probe is characterized in that the non-RNA coding region in Salmonella rDNA is used as the detection target gene, and the Salmonella rDNA spacer nucleic acid molecular probe is designed. Its structure is composed of 23 nucleotides, and its nucleotide The sequence is as follows: 5'CGAAGCATACATCAGTATGTTAG3'. 2. The application method of the Salmonella rDNA spacer nucleic acid molecular probe is characterized in that it is composed of 23 nucleotides, and its sequence is 5'CGAAGCATACATCAGTATGTTAG3'. When the probe is used for Salmonella inspection, the reference primer PCR method is used, and its application method It is to select more than one pair of primers with different specificities to perform PCR reaction on the same sample in the same test tube, and the PCR primer selection settings are as follows: ① Set a bacterial general-purpose PCR primer in the 3' terminal conservative region of the bacterial rDNA 16S rRNA coding region, and set another primer with Salmonella specificity in the spacer in the rDNA, that is, the Salmonella rDNA spacer between the nucleic acid molecular probe and the primer The distance is 207 nucleotides. ② Set another pair of bacterial rDNA universal primers in the 5' end conserved region of the bacterial rDNA 16S rRNA coding region, and the distance between them is 950 nucleotides.

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