CN114438177B - Method for rapidly quantifying ssDNA filamentous bacteriophage and application thereof - Google Patents

Method for rapidly quantifying ssDNA filamentous bacteriophage and application thereof Download PDF

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CN114438177B
CN114438177B CN202210137549.XA CN202210137549A CN114438177B CN 114438177 B CN114438177 B CN 114438177B CN 202210137549 A CN202210137549 A CN 202210137549A CN 114438177 B CN114438177 B CN 114438177B
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王晓雪
郭云学
林兼仲
汤开浩
高欣宇
古嘉瑜
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention discloses a method for quickly quantifying ssDNA filamentous bacteriophage and application thereof. The invention establishes a standard curve of Ct value (Y axis) obtained by qPCR measurement under each dilution gradient of Pf4 and Pf6 single-chain filamentous bacteriophage and the quantity (X axis) of Pf4 and Pf6 single-chain filamentous bacteriophage under each dilution gradient by combining a double-layer agar plate method and a qPCR method. The genome of the ssDNA of the filamentous phage can be quantified quickly and efficiently by combining the double-layer agar plate method with the qPCR determination method, and two ssDNA filamentous phage released simultaneously can be quantified and compared respectively.

Description

Method for rapidly quantifying ssDNA filamentous bacteriophage and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a method for quickly quantifying ssDNA filamentous bacteriophage and application thereof.
Background
Bacteriophages are viruses that infect bacteria, they are one of the important ways to mediate gene level transfer in microorganisms, and they are one of the prime movers to push the evolution of bacterial genomes. Bacteriophage has been widely used in molecular biology, antibacterial agents, and phage display, and phage therapy has recently become one of the hot methods for multi-drug resistant bacterial therapy. After entering the bacteria, the temperate phage "lurks" down, which is consistent with the replication pace of the host. When specific signals (such as antibiotics, ultraviolet rays and the like) transmitted from the outside are received, the latent people are aroused and ceaselessly 'rush' out of the host cells, so that the host bacteria die. The genetic material carried by these activated temperate bacteriophages includes single stranded DNA (ssDNA), double stranded DNA (dsDNA), single stranded RNA and double stranded RNA, with the majority of the bacteriophage genome being dsDNA. Filamentous phages, found half a century ago, are one of the simplest biological entities known today, with a genome size of approximately 7-12kb, as ssDNA.
Because the process of infecting bacteria by bacteriophage can cause the lysis of bacteria, inhibit the growth of bacteria, and generate plaques on agar plates containing specific host bacteria, one bacteriophage generally generates one plaque. Currently, the most popular method is to calculate the titer of the phage by the number of plaques appearing in a certain volume of phage culture solution, which is called double-layer agar plate method. However, the double-layer agar plate method takes a long time (as long as 18-24 hours) from plate preparation to infection experiment development until clear plaques are observed, and if different time points are required to be set in the same batch of samples for bacteriophage quantification, the double-layer plate preparation and spotting are required to be repeatedly carried out, which is time-consuming, labor-consuming and inefficient. The operation requires a relatively sterile environment, and if the phage contains two or more phage with highly similar genomes, the total amount of phage in the phage culture solution can only be quantified by a double-layer agar plate method, each phage cannot be quantified, and the method is not suitable for detection of multiple lysogenic phages. Another, relatively more adopted method is the Polymerase Chain Reaction (PCR) based fluorescent quantitative qPCR technique, which achieves effective quantification of the titer of dsDNA phage by designing primers for specific regions of the phage gene. Currently, the phage genomes reported to be quantified by qPCR methods are mostly dsDNA and RNA, relatively few for ssDNA genomes, and these reported qPCR methods require extraction of phage DNA followed by fluorescent quantitative PCR. In particular, it has now been found that filamentous phage are released without directly lysing the host and thus are less capable of plaque formation. Therefore, the development of an efficient and accurate method for quantifying the filamentous phage and distinguishing the proportion of each phage in the highly similar phage mixed culture solution has certain scientific significance for ssDNA phage-host interaction and release of different environmental factors and hosts to the filamentous phage, and also has important practical significance for researching pathogenicity of the filamentous phage and the hosts.
Disclosure of Invention
Aiming at the technical defects of the prior art, the invention aims to establish the qPCR method based on fluorescence quantification and capable of effectively amplifying ssDNA in filamentous phage so as to realize independent quantification of highly similar phage in host bacteria respectively, and also can quantify phage in a sample simultaneously containing similar phage respectively and determine the proportion of the phage.
It is a first object of the present invention to provide a method for rapid quantification of ssDNA filamentous phages comprising the steps of:
(1) Injecting pseudomonas aeruginosa bacterial liquid into a medical silicone tube of a micro-fluidic device, standing to enable bacteria to be adsorbed on the inner wall of the silicone tube, starting the micro-fluidic device, performing biofilm culture of the bacteria at room temperature by using an M9 culture medium, collecting effluent liquid, and filtering by using a microporous filter membrane to obtain sterile phage liquid;
(2) Constructing pseudomonas aeruginosa delta Pf4-Pf6 with Pf4 and Pf6 phage genes knocked out simultaneously, mixing pseudomonas aeruginosa delta Pf4-Pf6 bacterial liquid with an R-top culture medium, and paving the mixture on an LB (lysogeny broth) flat plate; taking the phage liquid obtained in the step (1) as a stock solution, performing gradient dilution, respectively spotting the stock solution and the diluent solution on an LB flat plate, performing overnight culture, and counting the total amount of phage in the stock solution and the diluent solution according to plaques;
(3) Adding DNase I and RNase I into the phage stock solution and the diluent respectively for processing, then carrying out qPCR detection on the phage stock solution and the diluent which are processed by DNase I and RNase I respectively by using primers pair pfkA-F/pfkA-R, pfiA-F/pfIA-R, and determining Ct values of Pf4 and Pf6 phages in the phage stock solution and the diluent;
(4) Determining the respective quantities of Pf4 and Pf6 phages in the phage stock solution and the dilution solution according to the total quantity of the phages measured in the step (2) and the proportion of Pf4 and Pf6 calculated from the Ct values of the Pf4 and Pf6 phages measured in the step (3);
(5) And (3) establishing a standard curve by taking the Ct value measured under each dilution concentration of the Pf4 or Pf6 bacteriophage as a vertical coordinate and the quantity of the bacteriophage measured under each dilution concentration as a horizontal coordinate, and obtaining the quantity of the corresponding single-type filamentous bacteriophage according to the Ct value measured by qPCR.
Further, in the step (1), the formula of the M9 culture medium is as follows: 47.8mM Na per liter of medium 2 HPO 4 、22mM KH 2 PO 4 、6.8mM NH 4 Cl、18.7mM NaCl、100μM CaCl 2 、2mM MgSO 4 0.1 percent of glucose, and the balance of water.
Further, in the step (1), the OD of the pseudomonas aeruginosa bacterial liquid 600 =1, the standing is standing for 1h; the flow rate of the microfluidic device is 0.1mL/min.
Further, in the step (2), the pseudomonas aeruginosa Δ Pf4-Pf6 is constructed by the following steps: firstly knocking out Pf4 prophage in pseudomonas aeruginosa, then knocking out Pf6 prophage from pseudomonas aeruginosa delta Pf4, and finally obtaining pseudomonas aeruginosa delta Pf4-Pf6, wherein the method specifically comprises the following steps:
s1: amplifying an upstream and a downstream region of Pf4 from the genome DNA of the pseudomonas aeruginosa PAO1 by PCR, and amplifying Pf4 upstream gene primer delta Pf4-F1:5'-GCCCCCGAGCTCGTTATTGGTCGTGGTTGCTTCCC-3' and Δ Pf4-R1:5'-GCCCCCGCTAGCGATCCCAATGCAAAAGCCCC-3', amplifying Pf4 downstream gene primer delta Pf4-F2:5'-GCCCCCGCTAGCTGGAGCGGGCGAAGGGAATCGAACCCTCG-3' and Δ Pf4-R2:5'-GCCCCCAAGTTCAGCCTGGACGAGCACGAATACC-3'; the gentamicin resistance gene cassette is amplified from the plasmid pPS856 by PCR, and a primer pEX18Gm-F of the gentamicin resistance gene cassette is amplified: 5'-AATCTTCTCTCATCCGCCAAAACA-3' and pEX18Gm-R:5'-CGCCCAATACGCAAACCGCCTCTC-3'; the three amplified fragments were then ligated into the suicide plasmid pEX18Ap using the ClonExpress II one-step cloning kit; adopting a sucrose resistance screening method, and obtaining an in-frame deletion mutant, namely pseudomonas aeruginosa delta Pf4, through homologous recombination;
s2: and (2) amplifying the upstream and downstream regions of Pf6 by using the genome DNA of pseudomonas aeruginosa delta Pf4 as a template, and amplifying Pf6 upstream gene primers delta Pf6-F1:5'-ACGACGGCCAGTGCCAAGCTTTCTGGCTACAAATTGACCACATG-3' and Δ Pf6-R1:5'-AGCGCAGCATTGTTTTGGTCGGCAGACTACAA-3', amplifying Pf6 downstream gene primer delta Pf6-F2:5'-GACCAAAACAATGCTGCGCTCTAACCGACT-3' and Δ Pf6-R2:5'-GGTACCCGGGGATCCTCTAGAGGGCAAGCCGGCGCGTTC-3'; the gentamicin resistance gene cassette was amplified from plasmid pPS856 by PCR; the three amplified fragments were then ligated into the suicide plasmid pEX18Ap using the ClonExpress II one-step cloning kit; adopting a sucrose resistance screening method, and obtaining an in-frame deletion mutant through homologous recombination, namely the pseudomonas aeruginosa delta Pf4-Pf6.
The correct mutants were confirmed by PCR and DNA sequencing.
Further, in the step (2), the formula of the R-top culture medium is as follows: each liter of the medium contained 1% tryptone, 0.1% yeast powder, 1% NaCl,0.8% agar, and the balance water.
Further, in the step (2), the volume ratio of the pseudomonas aeruginosa delta Pf4-Pf6 bacterial liquid to the R-top culture medium is 3; the overnight culture was 37 ℃ overnight culture.
Further, in the step (3), the final concentration of DNase I in the phage stock solution and the diluted solution is 0.02U/. Mu.L, the final concentration of RNase I in the phage stock solution and the diluted solution is 0.02U/. Mu.L, and the treatment is carried out at 37 ℃ for 1h.
Further, in the step (3), the sequence of the primer pair pfkA-F/pfkA-R is pfkA-F:5'-CGACCTGAAACCAACAAACG-3', pfkA-R:5'-TTAAACCATTGCTGAAAGGG-3'; the sequence of the primer pair pfiA-F/pfiA-R is as follows: pfiA-F:5'-ATGCGGCTGACCTGGATT-3', pfiA-R:5'-TGAGCGAACCTCCTGGAAA-3'.
Further, in the step (3), the reaction system of qPCR is 2 XChamQ SYRB qPCR Master Mix 10 μ L, primer F0.5 μ L, primer R0.5 μ L, DEPC water 8 μ L, and template DNA 1 μ L; the reaction sequence was 95 ℃ for 10min, followed by 95 ℃ for 15sec,60 ℃ for 1min for 40 cycles, and finally ended at 95 ℃ for 15sec,60 ℃ for 1min,95 ℃ for 15 sec.
It is a second object of the present invention to provide the use of the above-described method for rapid quantification of ssDNA filamentous bacteriophage in the quantification of ssDNA filamentous bacteriophage.
The ssDNA filamentous phage are released in large quantities during the biofilm formation of pseudomonas aeruginosa, and they can enhance the pathogenicity of bacteria and alter the immune response of eukaryotic hosts. Because the genome of pseudomonas aeruginosa carries two single-stranded filamentous phages Pf4 and Pf6 with highly conserved framework structures (the genome comparison and the electron microscope observation form of the two phages are shown in figure 1), and both of the phages can be released in a large amount in biofilm formation, but at present, no good method is available for distinguishing the phages, and the double-layer agar plate method which is most commonly used at present can only quantify the total amount of the phages. The method of quantifying phage by fluorescence quantification is mostly used for dsDNA and RNA, and few studies are used for ssDNA quantification. And the reported fluorescent quantitative method requires extraction of phage DNA followed by fluorescent quantitative PCR. The method of the invention does not need to extract the phage genome DNA, and can directly carry out the quantification of ssDNA phage particles by using the phage culture solution treated by DNaseI and RNaseI.
The invention aims to establish a detection method for simply and quickly distinguishing two single-chain filamentous phages Pf4 and Pf6 released in the process of forming a pseudomonas aeruginosa biofilm, and because the structural genes of the two single-chain filamentous phages are extremely similar and the main capsid proteins of the two single-chain filamentous phages cannot be distinguished, specific quantitative primers can be designed according to the fact that the genomes of the two single-chain filamentous phages carry respective accessory genes, so that the two single-chain filamentous phages can be distinguished efficiently and quickly. The method combines a double-layer agar plate method and a qPCR method to establish a standard curve of Ct value (Y axis) obtained by qPCR determination under each dilution gradient of single-stranded filamentous bacteriophage and the number (X axis) of single-stranded filamentous bacteriophage under each dilution gradient, combines the double-layer agar plate method and the qPCR determination method to quickly and efficiently quantify the ssDNA genome of the filamentous bacteriophage, can also respectively quantify and compare two ssDNA filamentous bacteriophage released at the same time, and does not detect the pollution of bacterial genome in the method established by the invention. More than half of clinically isolated pseudomonas aeruginosa contain filamentous phage, and a part of clinically isolated pseudomonas aeruginosa contain two or more filamentous phage, so the identification method based on qPCR provides a reliable detection method for the subsequent development of the role of the filamentous phage in biomembrane and bacterial pathogenicity processes.
Drawings
FIG. 1 shows comparative genomic (A) and electron microscopic morphology (B) of two single-stranded filamentous phages Pf4 and Pf6.
FIG. 2 is a diagram of a device for collecting phage by using a microfluidic culture biofilm.
FIG. 3 is a graph of primer amplification efficiency for quantification of single-stranded filamentous phages Pf4 and Pf6 using qPCR.
FIG. 4 is a standard curve established between the quantity and Ct value of Pf4 and Pf6 single-stranded filamentous phages released from biofilms.
FIG. 5 is a graph showing the results of verifying the release of Pf4 and Pf6 single-stranded filamentous phages on different days according to a standard curve.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
A method for rapid quantification of ssDNA filamentous bacteriophage comprising the steps of:
(1) The construction method comprises the steps of firstly knocking out Pf4 prophage in pseudomonas aeruginosa, then knocking out Pf6 prophage from the pseudomonas aeruginosa delta Pf4, and finally obtaining the pseudomonas aeruginosa delta Pf4-Pf6, wherein the construction method comprises the following steps: amplifying an upstream and a downstream region of Pf4 from the genome DNA of the pseudomonas aeruginosa PAO1 by PCR, and amplifying Pf4 upstream gene primer delta Pf4-F1:5'-GCCCCCGAGCTCGTTATTGGTCGTGGTTGCTTCCC-3' and Δ Pf4-R1:5'-GCCCCCGCTAGCGATCCCAATGCAAAAGCCCC-3', amplifying Pf4 downstream gene primer delta Pf4-F2:5'-GCCCCCGCTAGCTGGAGCGGGCGAAGGGAATCGAACCCTCG-3' and Δ Pf4-R2:5'-GCCCCCAAGTTCAGCCTGGACGAGCACGAATACC-3'; the gentamicin resistance gene cassette is amplified from the plasmid pPS856 by PCR, and a primer pEX18Gm-F of the gentamicin resistance gene cassette is amplified: 5'-AATCTTCTCTCATCCGCCAAAACA-3' and pEX18Gm-R:5'-CGCCCAATACGCAAACCGCCTCTC-3'; the three amplified fragments were then ligated into the suicide plasmid pEX18Ap using the ClonExpress II one-step cloning kit; adopting a sucrose resistance screening method, and obtaining an in-frame deletion mutant, namely pseudomonas aeruginosa delta Pf4, through homologous recombination; and (2) amplifying the upstream and downstream regions of Pf6 by using the genome DNA of pseudomonas aeruginosa delta Pf4 as a template, and amplifying Pf6 upstream gene primers delta Pf6-F1:5'-ACGACGGCCAGTGCCAAGCTTTCTGGCTACAAATTGACCACATG-3' and Δ Pf6-R1:5'-AGCGCAGCATTGTTTTGGTCGGCAGACTACAA-3', amplifying Pf6 downstream gene primer delta Pf6-F2:5'-GACCAAAACAATGCTGCGCTCTAACCGACT-3' and Δ Pf6-R2:5'-GGTACCCGGGGATCCTCTAGAGGGCAAGCCGGCGCGTTC-3'; amplifying a gentamicin resistance gene cassette from the plasmid pPS856 by PCR; the three amplified fragments were then ligated into the suicide plasmid pEX18Ap using the ClonExpress II one-step cloning kit; adopting a sucrose resistance screening method, and obtaining an in-frame deletion mutant through homologous recombination, namely the pseudomonas aeruginosa delta Pf4-Pf6. The correct mutants were confirmed by PCR and DNA sequencing.
(2) Inoculating pseudomonas aeruginosa PAO1 or pseudomonas aeruginosa delta Pf4-Pf6 on an LB (lysogeny broth) flat plate from a refrigerator at the temperature of-80 ℃, culturing overnight at the constant temperature of 37 ℃, selecting the pseudomonas aeruginosa PAO1 or the pseudomonas aeruginosa delta Pf4-Pf6, inoculating the bacteria to an LB liquid culture medium, and performing shake culture at the constant temperature of 37 ℃ to OD (origin of deficiency) 600 Is about 1.
(3) Microfluidic device for Pseudomonas aeruginosa PAO1 biofilm culture assembled as figure 2, aspirate 1mL OD 600 About 1 pseudomonas aeruginosa PAO1 inoculum is injected into a medical silica gel tube with the inner diameter of 3mm, and the medical silica gel tube is kept stand for 1h to ensure that bacteria are adsorbed on the inner wall of the silica gel tube. Starting the microfluidic device, and controlling the M9 culture medium (formula: 47.8mM Na in each liter of culture medium) 2 HPO 4 ,22mM KH 2 PO 4 ,6.8mM NH 4 Cl,18.7mM NaCl,100μM CaCl 2 ,2mM MgSO 4 0.1% glucose, and the balance water; preparation: dissolving the components in water according to the formula amount, and sterilizingObtained) the flow rate is 0.1mL/min, the biomembrane of the pseudomonas aeruginosa PAO1 is cultured at room temperature, the effluent is collected every day, and then the effluent is filtered by a 0.22 mu m millipore filter membrane and collected to obtain the bacteriophage liquid without bacteria.
(4) Using a double agar plate method, 3mL of OD was added 600 Pseudomonas aeruginosa delta Pf4-Pf6 bacterial solution of about 1 is mixed with 10mL of R-top culture medium (the temperature is about 55 ℃) (formula: 1% tryptone, 0.1% yeast powder, 1% NaCl,0.8% agar and the balance of water are contained in each liter of culture medium; preparation: dissolving the components in water according to formula amount and sterilizing the components) and then is flatly paved on an LB plate, and the mixture is kept still for 10min on an ultra-clean bench and is used for phage spotting operation. Taking the phage liquid obtained in step (3) every day as stock solution, and adding 10 g of the stock solution 1 ～10 8 And (4) performing gradient dilution, sucking 5 mu L of stock solution and diluent solution to a LB plate, performing constant temperature overnight culture at 37 ℃, and counting the total quantity of the phage in the stock solution and the diluent solution according to plaques.
(5) Add DNase I and RNase I to 100 μ L phage stock and dilution respectively to the final concentration of 0.02U/. Mu.L, then treat at 37 ℃ for 1h to remove DNA and RNA impurities secreted by bacteria or broken down by death, ssDNA filamentous phage because of the protein shell protection, whose genetic material ssDNA is not hydrolyzed by DNase I. Specific quantitative primers of Pf4 and Pf6 ssDNA filamentous phage are designed, and meanwhile, a single copy gene gyrB of genome is used as a reference gene to design the quantitative primers, whether the DNA of bacterial genome is completely treated by DNase I is detected (Table 1), and the result shows that the pollution of bacterial genome is not detected in the method established by the invention, and the amplification efficiency of the checking primers is shown in figure 3. Respectively taking 1 mu L of phage stock solution and diluent which are treated by DNase I and RNase I to carry out qPCR detection, wherein a qPCR reaction system is 2 XChamQ SYRB qPCR Master Mix 10 mu L, a primer F0.5 mu L, a primer R0.5 mu L, DEPC water 8 mu L and template DNA 1 mu L; the reaction sequence was 95 ℃ for 10min, then 95 ℃ for 15sec, then 60 ℃ for 1min for 40 cycles, and finally 95 ℃ for 15sec,60 ℃ for 1min,95 ℃ for 15 sec. Ct values of Pf4 and Pf6 single-stranded filamentous phages were determined in phage stocks and dilutions.
TABLE 1 primers used in the present invention
Figure BDA0003505543720000091
(6) Determining the respective quantities of Pf4 and Pf6 single-chain filamentous phages in the phage stock solution and the dilution solution according to the total quantity of the phages measured in the step (4) and the ratio of Pf4 and Pf6 calculated from the Ct values of the Pf4 and Pf6 single-chain filamentous phages measured in the step (5);
calculating the formula:
total phage/ml = number of plaques x dilution multiple x 200 under appropriate dilution gradient
If Ct is Pf6 -Ct Pf4 >Total of 0,Pf6 = total number of phages x 2^ - (Ct) Pf6 -Ct Pf4 )
Total of Pf4 = total number of phages-number of Pf6
If Ct is Pf4 -Ct Pf6 >Total of 0,Pf4 = total number of phages x 2^ - (Ct) Pf4 -Ct Pf6 )
The total number of Pf6 = the total number of phages-the number of Pf 4.
(7) Taking the Ct value measured at each dilution concentration of the Pf4 or Pf6 single-stranded filamentous bacteriophage as the ordinate (Y axis) and the number of the bacteriophage measured at each dilution concentration as the abscissa (X axis), a standard curve is established, and the number of the corresponding single-type filamentous bacteriophage is obtained according to the Ct value measured by qPCR (FIG. 4).
(8) To verify the feasibility of the method of the present invention, we performed qPCR assay on phage released by pseudomonas aeruginosa PAO1 biofilm on different days using the above method to obtain Ct values of Pf4 and Pf6 single-stranded filamentous phage on different days, and calculated the number of Pf4 and Pf6 single-stranded filamentous phage on different days according to the standard curve of fig. 4. The number of days was plotted on the abscissa (X-axis) and the number of phage calculated on the ordinate (Y-axis) (FIG. 5).
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims (7)

1. A method for rapid quantification of ssDNA filamentous bacteriophage for non-disease diagnostic and therapeutic purposes, comprising the steps of:
(1) Injecting pseudomonas aeruginosa bacterial liquid into a medical silicone tube of a micro-fluidic device, standing to enable bacteria to be adsorbed on the inner wall of the silicone tube, starting the micro-fluidic device, performing biofilm culture of the bacteria at room temperature by using an M9 culture medium, collecting effluent liquid, and filtering by using a microporous filter membrane to obtain sterile phage liquid;
(2) Constructing pseudomonas aeruginosa delta Pf4-Pf6 with Pf4 and Pf6 phage genes knocked out simultaneously, mixing pseudomonas aeruginosa delta Pf4-Pf6 bacterial liquid with an R-top culture medium, and paving the mixture on an LB (lysogeny broth) flat plate; taking the phage liquid obtained in the step (1) as a stock solution, performing gradient dilution, respectively spotting the stock solution and the diluent solution on an LB flat plate, performing overnight culture, and counting the total amount of phage in the stock solution and the diluent solution according to plaques;
(3) Adding DNase I and RNase I into the phage stock solution and the diluent respectively for treatment, and then carrying out qPCR detection on the phage stock solution and the diluent treated by the DNase I and the RNase I by using a primer pair of pfkA-F/pfkA-R, pfiA-F/pfiA-R, wherein the sequence of the primer pair of pfkA-F/pfkA-R is pfkA-F:5'-CGACCTGAAACCAACAAACG-3', pfkA-R:5'-TTAAACCATTGCTGAAAGGG-3'; the sequence of the primer pair pfiA-F/pfiA-R is as follows: pfiA-F:5'-ATGCGGCTGACCTGGATT-3', pfiA-R:5'-TGAGCGAACCTCCTGGAAA-3', determining the Ct values of Pf4 and Pf6 phages in phage stock solution and dilution solution;
(4) Determining the respective quantities of Pf4 and Pf6 phages in the phage stock solution and the dilution solution according to the total quantity of the phages measured in the step (2) and the proportion of Pf4 and Pf6 calculated from the Ct values of the Pf4 and Pf6 phages measured in the step (3);
(5) Establishing a standard curve by taking the Ct value measured under each dilution concentration of Pf4 or Pf6 bacteriophage as a vertical coordinate and the quantity of the bacteriophage measured under each dilution concentration as a horizontal coordinate, and obtaining the quantity of the corresponding single type filamentous bacteriophage according to the Ct value measured by qPCR;
the formula of the R-top culture medium is as follows: per liter of the medium containing 1% tryptone, 0.1% yeast powder, 1% NaCl,0.8% agar, the balance water;
the pseudomonas aeruginosa delta Pf4-Pf6 is constructed by the following steps:
s1: amplifying an upstream and a downstream region of Pf4 from the genome DNA of the pseudomonas aeruginosa PAO1 by PCR, and amplifying Pf4 upstream gene primer delta Pf4-F1:5'-GCCCCCGAGCTCGTTATTGGTCGTGGTTGCTTCCC-3' and Δ Pf4-R1:5'-GCCCCCGCTAGCGATCCCAATGCAAAAGCCCC-3', amplifying Pf4 downstream gene primer delta Pf4-F2:5'-GCCCCCGCTAGCTGGAGCGGGCGAAGGGAATCGAACCCTCG-3' and Δ Pf4-R2:5'-GCCCCCAAGTTCAGCCTGGACGAGCACGAATACC-3'; the gentamicin resistance gene cassette is amplified from the plasmid pPS856 by PCR, and a primer pEX18Gm-F of the gentamicin resistance gene cassette is amplified: 5'-AATCTTCTCTCATCCGCCAAAACA-3' and pEX18Gm-R:5'-CGCCCAATACGCAAACCGCCTCTC-3'; the three amplified fragments were then ligated into the suicide plasmid pEX18Ap using the ClonExpress II one-step cloning kit; adopting a sucrose resistance screening method, and obtaining an in-frame deletion mutant, namely pseudomonas aeruginosa delta Pf4, through homologous recombination;
s2: and (2) amplifying the upstream and downstream regions of Pf6 by using the genome DNA of pseudomonas aeruginosa delta Pf4 as a template, and amplifying Pf6 upstream gene primers delta Pf6-F1:5'-ACGACGGCCAGTGCCAAGCTTTCTGGCTACAAATTGACCACATG-3' and Δ Pf6-R1:5'-AGCGCAGCATTGTTTTGGTCGGCAGACTACAA-3', amplifying Pf6 downstream gene primer Δ Pf6-F2:5'-GACCAAAACAATGCTGCGCTCTAACCGACT-3' and Δ Pf6-R2:5'-GGTACCCGGGGATCCTCTAGAGGGCAAGCCGGCGCGTTC-3'; amplifying a gentamicin resistance gene cassette from the plasmid pPS856 by PCR; the three amplified fragments were then ligated into the suicide plasmid pEX18Ap using the ClonExpress II one-step cloning kit; adopting a sucrose resistance screening method, and obtaining an in-frame deletion mutant through homologous recombination, namely the pseudomonas aeruginosa delta Pf4-Pf6.
2. The method of rapidly quantifying ssDNA filamentous phages according to claim 1, wherein in step (1),the formula of the M9 culture medium is as follows: 47.8mM Na per liter of medium 2 HPO 4 、22mM KH 2 PO 4 、6.8mM NH 4 Cl、18.7mM NaCl、100μM CaCl 2 、2mM MgSO 4 0.1 percent of glucose and the balance of water.
3. The method according to claim 1, wherein, in step (1), the OD of the bacterial solution of Pseudomonas aeruginosa is determined by the concentration of ssDNA filamentous phage 600 =1, wherein the standing is 1h; the flow rate of the microfluidic device is 0.1mL/min.
4. The method for rapid quantification of ssDNA filamentous phage according to claim 1, wherein in step (2), the volume ratio of the P.aeruginosa Δ Pf4-Pf6 bacterial liquid to the R-top medium is 3; the overnight culture was 37 ℃ overnight culture.
5. The method for rapid quantification of ssDNA filamentous bacteriophage according to claim 1, wherein in step (3), said DNase I is present in a final concentration of 0.02U/. Mu.L in phage stock solution and dilution, said RNase I is present in a final concentration of 0.02U/. Mu.L in phage stock solution and dilution, and said treating is carried out at 37 ℃ for 1h.
6. The method according to claim 1, wherein in step (3), the qPCR reaction system is 2 XChamQ SYRB qPCR Master Mix 10. Mu.L, primer F0.5. Mu.L, primer R0.5. Mu.L, DEPC water 8. Mu.L, and template DNA 1. Mu.L; the reaction sequence was 95 ℃ for 10min, followed by 95 ℃ for 15sec,60 ℃ for 1min for 40 cycles, and finally ended at 95 ℃ for 15sec,60 ℃ for 1min,95 ℃ for 15 sec.
7. Use of the method of rapidly quantifying ssDNA filamentous phages according to any of claims 1-6 for the quantification of ssDNA filamentous phages Pf4 and Pf6 for non-disease diagnostic and therapeutic purposes.
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