CN110819699A - A Quantitative Detection Method of Human Fecal Indicators in Water Environment - Google Patents

Abstract

The invention relates to a quantitative detection method for human feces indicators in a water environment. Including: selecting human fecal DNA as a template, using PCR to amplify the target gene CPQ_056 fragment; cutting the gel to recover and purify the amplified target gene, connecting the gene to the vector pTOPO-Blunt, then transforming competent Escherichia coli cells, and selecting positive clones Use agarose gel electrophoresis to detect the quality of the expanded culture bacterial liquid, select the successfully cloned bacterial liquid to extract the plasmid, detect the plasmid concentration, convert the copy number of the target gene carried by the plasmid, and dilute it by 10 times as a dilution gradient, as Standard substance; perform quantitative PCR amplification on the standard substance to make a standard curve. The water sample DNA was then amplified by quantitative PCR to finally obtain the copy number of the human fecal indicator gene in the water environment. The invention does not depend on microbial culture, has simple operation, high detection speed, high sensitivity, and is more intuitive in quantity.

Description

A Quantitative Detection Method of Human Fecal Indicators in Water Environment

technical field

The invention belongs to the field of biotechnology, and in particular relates to a quantitative detection method for human feces indicators in a water environment.

Background technique

Environmental pollution and public health problems caused by human fecal discharge have always attracted much attention. Human feces contain a large number of pollutants such as pathogenic bacteria and drug-resistant genes, which are discharged into the environment through untreated or partially treated sewage, resulting in pollution of many environmental waters. Currently, water quality monitoring mainly uses fecal bacteria as indicators of fecal contamination in the water environment, but these indicators cannot differentiate between human and animal feces, many previously developed indicators of human fecal contamination lack sufficient sensitivity to be reliably detected in environmental waters, or Not well correlated with viral pathogens.

In recent years, researchers discovered a new bacteriophage crAssphage through metagenomic data mining, and reported that it is abundant in human feces and is closely related to human fecal pollution. Therefore, it is of great significance to use biotechnology to find a highly human-specific, abundant in sewage, which can efficiently determine the copy number of the indicator in different water environments, and then detect the discharge of human feces in the water environment.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a quantitative detection method of human feces indicator in water environment with high human specificity and efficient determination of the copy number of target gene in different water environments.

The technical scheme adopted in the present invention is: a quantitative detection method of human feces indicator in a water environment, comprising the following steps:

(1) Generating the standard curve of the target gene: take the CPQ_056 gene as the target gene, extract the CPQ_056 gene fragment from human feces, take the logarithm of the copy number of the target gene CPQ_056 as the abscissa, and the Ct value as the ordinate to generate the CPQ_056 target gene standard curve; specifically:

1) Extracting the DNA of human feces; using human feces DNA as a template, using forward primer F-primer and reverse primer R-primer as specific primers and probes to carry out qualitative PCR amplification to amplify the fragment of the target gene CPQ_056;

The sequences of the forward primer F-primer, the reverse primer R-primer and the probe are:

F-primer: CAGAAGTACAAACTCCTAAAAAACGTAGAG

R-primer: GATGACCAATAAACAAGCCATTAGC

Probe: [FAM]AATAACGATTTACGTGATGTAAC[MGB]

The qualitative PCR amplification, the qualitative PCR amplification system is, the volume of the reaction system is 25 μL, including:

Qualitative PCR amplification reaction program: 95°C 5min, 95°C 30s, 59°C 30s 40 cycles, 72°C 30s, 72°C 4min.

2) The fragment of the target gene CPQ_056 after the recovery, purification and amplification was performed by cutting the gel, and the target gene CPQ_056 was connected to the pTOPO-Blunt carrier, and then transferred into the competent Escherichia coli. Then select positive clones and expand the culture with LB liquid medium;

3) use agarose gel electrophoresis to detect the quality of the expanded culture bacterial liquid, select the successful bacterial liquid to clone to extract plasmid, detect the plasmid concentration, convert the copy number of the target gene CPQ_056 carried by the plasmid by formula (1), and press 10 times For serial dilution, prepare bacterial solutions containing different copy numbers;

Copies/μL=[x/(a+b)×660]×10 ⁻⁹ ×6.02×10 ²³ (I)

Among them, Copies/μL: the absolute copy number of the target gene of CPQ_056 carried by each μL of plasmid solution;

X: is the plasmid concentration (ng/μL);

a: is the length of the expression vector pTOPO-Blunt, which is 1865bp;

b: is the length of the CPQ_056 gene, which is 126bp

4) carrying out quantitative PCR amplification with bacterial liquids containing different copy numbers, respectively, to obtain Ct values; take the logarithm of the copy number of the CPQ_056 target gene as the abscissa, and Ct be the value ordinate, and generate the standard curve of the CPQ_056 target gene;

The quantitative PCR amplification system is: the volume of the reaction system is 20 μL, including:

Quantitative PCR amplification reaction program: 94°C for 30s, 94°C for 5s, 59°C for 15s for 40 cycles, and 72°C for 15s.

(2) Sample detection: take the water sample to be tested, extract the DNA of the water sample; use the water sample DNA as a template, carry out quantitative PCR amplification with specific primers and probes, and obtain the Ct value;

The specific primers are forward primer F-primer and reverse primer R-primer, and the sequences of forward primer F-primer, reverse primer R-primer and probe are:

F-primer: CAGAAGTACAAACTCCTAAAAAACGTAGAG

R-primer: GATGACCAATAAACAAGCCATTAGC

Probe: [FAM]AATAACGATTTACGTGATGTAAC[MGB]

The quantitative PCR amplification system is: the volume of the reaction system is 20 μL, including:

Quantitative PCR amplification reaction program: 94°C for 30s, 94°C for 5s, 59°C for 15s for 40 cycles, and 72°C for 15s

(3) Calculation: According to the measured Ct value and the standard curve of the CPQ_056 target gene, the copy number of the CPQ_056 target gene in the water sample to be tested is obtained.

The beneficial effects of the present invention are:

1. In recent years, researchers discovered a new bacteriophage crAssphage through metagenomic data mining, and reported that it is abundant in human feces and is closely related to human fecal pollution. Based on the phage crAssphage, two novel human sewage-related source-tracking qPCR markers, CPQ_056 and CPQ_064, were developed. CPQ_056 has a high degree of human specificity and is abundant in sewage. By detecting the copy number of CPQ_056 in the water environment, the invention can quickly detect the discharge of human feces in the water environment.

2. The present invention uses human fecal samples to accurately and rapidly prepare the plasmid standard of the target gene, and performs absolute quantification of the CPQ_056 gene in the sample by preparing a standard curve. Plasmid standards can be stored at -20°C. The method has the advantages of simple operation, high detection speed for samples, high sensitivity of detection results, and more intuitive quantitative results, so that the finally obtained quantitative results are more comprehensive and credible.

Description of drawings

Figure 1 is a structural diagram of the expression vector pTOPO-Blunt.

Figure 2 is a gel electrophoresis image of the target gene CPQ_056.

Figure 3a is the Ct value of the target gene of CPQ_056 with different copy numbers of the standard.

Figure 3b is a standard curve diagram of the target gene of standard CPQ_056.

Detailed ways

Example 1

The quantitative detection method of human fecal indicator in water environment includes the following steps:

(1) Standard curve drawing of target gene CPQ_056

1) Take human feces, and extract the DNA of human feces with PowerFecal DNA Isolation Kit (MoBio, USA).

The DNA extracted from human feces was used as the template, and the forward primer F-primer and the reverse primer R-primer were used as specific primers and probes for qualitative PCR amplification to amplify the fragment of the target gene CPQ_056.

The sequences of the forward primer F-primer, the reverse primer R-primer and the probe are:

F-primer: CAGAAGTACAAACTCCTAAAAAACGTAGAG

R-primer: GATGACCAATAAACAAGCCATTAGC

Probe: [FAM]AATAACGATTTACGTGATGTAAC[MGB]

The qualitative PCR amplification system is: the volume of the reaction system is 25 μL, including:

The qualitative PCR amplification program was as follows: 95°C for 5 min, 40 cycles of 95°C for 30s, 59°C for 30s, 72°C for 30s, and 72°C for 4min.

The agarose gel electrophoresis of the PCR amplification product is shown in Figure 2. It can be seen from Figure 2 that all three parallel samples detected a band at a length of 126bp, indicating that the PCR amplification experiment was successful and the product was the desired target gene fragment CPQ_056 .

2) Purify the PCR amplification product using AXYPREP DNA Gel Extraction Kit. Using the complementarity of A-T bases, the target gene CPQ_056 fragment was ligated to the vector pTOPO-Blunt (as shown in Figure 1) to form a recombinant plasmid containing the target gene CPQ_056. Specifically: add 4 μL of the pure PCR amplification product recovered from the gel and 1 μL of T-vector (pTOPO-Blunt Simple Cloning Vector) into a sterile centrifuge tube, shake and mix, and react at room temperature (20°C-37°C) for 5 minutes, the target gene CPQ_056 was connected to the vector pTOPO-Blunt, and the centrifuge tube was placed on ice immediately after the reaction.

3) Transformation. The recombinant plasmid containing the target gene CPQ_056 was transformed into E. coli competent cells. Specifically: add the recombinant plasmid containing the target gene CPQ_056 to 50 μL of E. coli competent cells that have just been thawed, flick and mix, and place it on ice for 20-30 minutes; immediately after the ice bath, place it in a water bath at 42°C Medium heat shock for 30 s, and then immediately placed on ice for 2 min to transfer the constructed recombinant plasmid into competent cells. Then, add 250 μL of sterilized fresh LB liquid medium to this bacterial solution, incubate at 200 rpm in a shaking incubator at 37°C for 1 hour to recover competent cells; after recovery, take 100 μL of bacterial solution and spread evenly on The prepared LB solid plates containing 8 μL of 500 mM IPTG and 40 μL of 20 mg/mL X-gal were incubated for 12-16 h in a constant temperature incubator at 37 °C. After the cultivation, under sterile conditions, pick white plaques and inoculate them into LB liquid medium containing 100 μg/mL ampicillin, and then place them in a shaker incubator with a rotating speed of 200 rpm, and cultivate at 37 °C. 12-16h to expand the culture of Escherichia coli containing the target gene of CPQ_056.

4) Sequencing identification of positive recombinants. After overnight culture, the plasmids in the positive clones were extracted with a plasmid mini-kit (OMEGA), and then the extracted plasmid DNA was used as a template, and the primers M13F (TGTAAAACGACGGCCAGT) and M13R (CAGGAAACAGCTATGACC) were used in the carrier kit. ) Amplify and sequence the fragments inserted in the vector (completed by Beijing Aoke Dingsheng Biotechnology Co., Ltd.), find the fragments inserted in the multi-cloning site, and then obtain the inserted sequence on the BLAST of the NCBI website (http: //www.ncbi.nlm.nih.gov/blast/), and check whether it is consistent with the target gene sequence. After comparison, it is finally confirmed that the standard plasmid containing the target gene CPQ_056 fragment has been successfully prepared.

5) Select the cloned bacterial liquid to extract the plasmid, use a nucleic acid protein analyzer to detect the concentration of the extracted plasmid, and dilute it by 10-fold dilution gradient. A total of 8 gradient dilutions are used as the standard, and the concentrations from high to low are: 15ng /μL, 1.5ng/μL, 1.5× ^10-1 ng/μL, 1.5× ^10-2 ng/μL, 1.5× ^10-3 ng/μL, 1.5× ^10-4 ng/μL, 1.5× ^10-5 ng /μL, 1.5×10 ^-6 ng/μL, according to formula (I), convert the copy number of the target gene of CPQ_056 carried by the plasmid, and prepare the bacterial liquid containing different copy numbers.

In the present invention, the formula for converting the plasmid concentration into the copy number of the target gene of CPQ_056 carried by each μL of the plasmid solution is:

Copies/μL=[x/(a+b)×660]×10 ⁻⁹ ×6.02×10 ²³ (I)

Among them, Copies/μL: the number of copies of the target gene of CPQ_056 carried by each μL of plasmid solution;

X: is the plasmid concentration (ng/μL);

a: is the length of the expression vector pTOPO-Blunt (1865bp);

b: is the length of the CPQ_056 gene (126bp)

6) carrying out quantitative PCR amplification with bacterial liquids containing different copy numbers, respectively, to obtain Ct value; take the logarithm of the copy number of the CPQ_056 target gene as the abscissa, and the ordinate be the Ct value, and make the standard curve of the CPQ_056 target gene;

The quantitative PCR amplification system is: the volume of the reaction system is 20 μL, including:

Quantitative PCR amplification reaction program: 94°C for 30s, 94°C for 5s, 59°C for 15s for 40 cycles, and 72°C for 15s.

The results are shown in Figure 3a and Figure 3b, the measured standard curve of CPQ_056 is y=-3.205x+44.427

The above x is the log10 copy number of the target gene, and y is the critical cycle number, that is, the Ct value reading of the quantitative PCR instrument. From the above curve, the linear relationship between the Ct value and the copy number of the target gene is very strong, the R ² is 0.996, and the amplification efficiency is 105.1%.

(2) Sample detection

1) The water sample was filtered and retained through a 0.22 μm water-based microporous filtration membrane (Jinteng, Tianjin), and the total DNA of the water sample was extracted using the ENZA Water DNA kit (Omega, USA);

2) Using the water sample DNA as a template, using specific primers and probes to detect the target gene CPQ_056 in the sample by SmartChip qPCR (Wafergen, USA) high-throughput fluorescence quantitative reaction system, and obtain the corresponding Ct value.

Use forward primer F-primer and reverse primer R-primer as specific primers. The sequences of the forward primer F-primer, the reverse primer R-primer and the probe are:

F-primer: CAGAAGTACAAACTCCTAAAAAACGTAGAG

R-primer: GATGACCAATAAACAAGCCATTAGC.

Probe: [FAM]AATAACGATTTACGTGATGTAAC[MGB]

The quantitative PCR amplification system is: the volume of the reaction system is 20 μL, including:

Quantitative PCR amplification reaction program: 94°C for 30s, 94°C for 5s, 59°C for 15s for 40 cycles, and 72°C for 15s.

(3) Calculation: According to the measured Ct value and the standard curve of the CPQ_056 target gene, the copy number of the CPQ_056 target gene in the water sample to be tested is obtained.

(4) Determination of actual samples

The influent and effluent samples of a sewage treatment plant in Changzhou City and the water samples of the sewage receiving river were taken, and the method of step (2) was used to calculate the copy number of the target gene of CPQ_056 in the influent water, which was 9.44×10 ⁹ copies/L. The number of copies was 3.03×10 ⁵ copies/L, 2.19×10 ⁶ copies/L in the upper reaches of the Sewage River, 2.81×10 ⁵ copies/L at the sewage outlet, 3.72×10 ⁶ copies/L at 200m downstream of the Sewage River, and 2.81×10 5 copies/L at the sewage outlet. 2.15×10 ⁶ copies/L at 400m downstream. It can be seen that the influent indicator gene of the sewage plant is the highest, which is the sink of fecal discharge. After treatment, the indicator gene has been reduced by 4 orders of magnitude. The indicator genes of the upstream and downstream of the sewage receiving river are relatively high, indicating that the surrounding human activities have caused serious water pollution. The effluent abundance of the sewage treatment plant is similar and slightly lower than that of the sewage receiving river, indicating that the effluent standard of the sewage treatment plant is relatively high.

Claims (4)

1. a quantitative detection method of human excrement indicator in a water environment, is characterized in that, comprises the steps: (1) Generating the standard curve of the target gene: take the CPQ_056 gene as the target gene, extract the CPQ_056 gene fragment from human feces, take the logarithm of the copy number of the target gene CPQ_056 as the abscissa, and the Ct value as the ordinate to generate the CPQ_056 target gene standard curve; (2) Sample detection: take the water sample to be tested, extract the DNA of the water sample; use the water sample DNA as a template, carry out quantitative PCR amplification with specific primers and probes, and obtain the Ct value; (3) Calculation: According to the measured Ct value and the standard curve of the CPQ_056 target gene, the copy number of the CPQ_056 target gene in the water sample to be tested is obtained. 2. quantitative detection method according to claim 1, is characterized in that, in step (2), described specific primer is forward primer F-primer and reverse primer R-primer, forward primer F-primer, The sequences of the reverse primer R-primer and probe are: F-primer: CAGAAGTACAAACTCCTAAAAAACGTAGAG R-primer: GATGACCAATAAACAAGCCATTAGC. Probe: [FAM]AATAACGATTTACGTGATGTAAC[MGB] 3. quantitative detection method according to claim 2, is characterized in that, in step (2), described quantitative PCR amplification is, The quantitative PCR amplification system is: the volume of the reaction system is 20 μL, including:

Quantitative PCR amplification reaction program: 94°C for 30s, 94°C for 5s, 59°C for 15s for 40 cycles, and 72°C for 15s. 4. quantitative detection method according to claim 3, is characterized in that, step (1) making target gene standard curve, comprises the steps: 1) extracting the DNA of human feces; using human feces DNA as a template, using forward primer F-primer and reverse primer R-primer as specific primers and probes to carry out qualitative PCR amplification to amplify the fragment of the target gene CPQ_056; The sequences of the forward primer F-primer, the reverse primer R-primer and the probe are: F-primer: CAGAAGTACAAACTCCTAAAAAACGTAGAG R-primer: GATGACCAATAAACAAGCCATTAGC Probe: [FAM]AATAACGATTTACGTGATGTAAC[MGB] The qualitative PCR amplification, the qualitative PCR amplification system is, the volume of the reaction system is 25 μL, including:

Qualitative PCR amplification reaction program: 95°C for 5 min, 95°C for 30s, 59°C for 30s for 40 cycles, 72°C for 30s, 72°C for 4min. 2) The fragment of the target gene CPQ_056 after the recovery, purification and amplification was performed by cutting the gel, the target gene CPQ_056 was connected on the pTOPO-Blunt carrier, and then transferred into the competent Escherichia coli. Then select positive clones and expand the culture with LB liquid medium; 3) use agarose gel electrophoresis to detect the quality of the expanded culture bacterial liquid, select the successful bacterial liquid to clone to extract plasmid, detect the plasmid concentration, convert the copy number of the target gene CPQ_056 carried by the plasmid by formula (1), and press 10 times For serial dilution, prepare bacterial solutions containing different copy numbers; Copies/μL=[x/(a+b)×660]×10 ⁻⁹ ×6.02×10 ²³ (I) Among them, Copies/μL: the absolute copy number of the target gene of CPQ_056 carried by each μL of plasmid solution; X: is the plasmid concentration (ng/μL); a: is the length of the expression vector pTOPO-Blunt, which is 1865bp; b: is the length of the CPQ_056 gene, which is 126bp 4) carrying out quantitative PCR amplification with bacterial liquids containing different copy numbers, respectively, to obtain Ct values; take the logarithm of the copy number of the CPQ_056 target gene as the abscissa, and Ct be the value ordinate, and generate the standard curve of the CPQ_056 target gene; The quantitative PCR amplification system is: the volume of the reaction system is 20 μL, including:

Quantitative PCR amplification reaction program: 94°C for 30s, 94°C for 5s, 59°C for 15s for 40 cycles, and 72°C for 15s.

Images