Detection method of escherichia coli RNA (Ribonucleic Acid) and application thereof

Abstract

The invention relates to a detection method of escherichia coli RNA (Ribonucleic Acid) and application thereof. Specifically, the invention discloses a quantitative method for rapidly detecting RNA in escherichia coli in a sample. According to the invention, total RNA in Escherichia coli is taken as a standard substance, a qPCR method is used, and the content of Escherichia coli RNA in a sample is quantitatively detected and analyzed, so that whether the sample is polluted by Escherichia coli or not can be determined, the pollution degree can be evaluated, or the residual amount of Escherichia coli RNA in the sample can be determined. The detection sensitivity is high, the detection limit can reach 0.01 pg / microliter, the escherichia coli can be effectively distinguished from RNA of other types and sources, and the specificity is high. The method is wide in application range, simple and rapid to operate and low in cost.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically relates to a detection method for Escherichia coli RNA and an application thereof. Background Art

[0002] In the fields of environment, water and food hygiene, Escherichia coli can be used as an indicator bacteria for health quality monitoring. Under poor environmental hygiene conditions, Escherichia coli may contaminate agricultural water and then contaminate agricultural products, causing outbreaks of food safety diseases. Therefore, Escherichia coli needs to be monitored. Generally, methods such as coliform group counting and rapid isolation and culture are used. However, there are defects such as low detection sensitivity, inability to accurately quantify, and long detection cycle (lasting several days).

[0003] In the fields of biopharmaceuticals and genetic engineering, Escherichia coli is the most widely used host microorganism; after the exogenous gene is loaded into the host, a large amount of target product can be obtained through the rapid reproduction of the host. In order to obtain a safe final product, the preliminary target product needs to be strictly inspected after purification to ensure that various impurities remaining in the host, including endotoxins, DNA, RNA, proteins, etc., have been effectively removed. At present, the regulations of various countries have clear requirements for host residual DNA, proteins and / or endotoxins, but there are no definite requirements for host residual RNA, so there is little research in this aspect in the prior art. There are currently methods based on spectrophotometry and fluorescent dyes to detect host residual RNA, but these methods have the following defects: the ultraviolet absorption spectrophotometer method can detect the lowest content of 2ng / μl, and the fluorescent dye method can detect the lowest limit of 2pg / μl, but both cannot distinguish RNA from DNA, let alone Escherichia coli RNA from RNA of other species. At present, there is still a lack of technical solutions that can effectively and quickly detect host residual RNA in this field.

[0004] In summary, there is an urgent need to develop an efficient, rapid and quantitative method for detecting Escherichia coli RNA in related fields. Summary of the invention

[0005] The purpose of the present invention is to provide a method for efficiently, quickly and quantitatively detecting Escherichia coli RNA and its application.

[0006] In a first aspect of the present invention, a primer pair for detecting Escherichia coli RNA is provided, the primer pair comprising a forward primer and a reverse primer, and the primer pair is selected from the following group:

[0007] (a) Primer pair 1, wherein the sequence of the forward primer is shown in SEQ ID NO: 1, and the sequence of the reverse primer is shown in SEQ ID NO: 19;

[0008] (b) Primer pair 2, wherein the sequence of the forward primer is shown in SEQ ID NO: 2, and the sequence of the reverse primer is shown in SEQ ID NO: 20;

[0009] (c) Primer pair 3, wherein the sequence of the forward primer is shown in SEQ ID NO: 3, and the sequence of the reverse primer is shown in SEQ ID NO: 21;

[0010] or a combination thereof.

[0011] In another preferred embodiment, the primer pair further comprises:

[0012] Primer pair 4, wherein the sequence of the forward primer is shown in SEQ ID NO:4, and the sequence of the reverse primer is shown in SEQ ID NO:22;

[0013] Primer pair 5, wherein the sequence of the forward primer is shown in SEQ ID NO:5, and the sequence of the reverse primer is shown in SEQ ID NO:23;

[0014] Primer pair 6, wherein the sequence of the forward primer is shown in SEQ ID NO: 6, and the sequence of the reverse primer is shown in SEQ ID NO: 24;

[0015] Primer pair 7, wherein the sequence of the forward primer is shown in SEQ ID NO:7, and the sequence of the reverse primer is shown in SEQ ID NO:25;

[0016] Primer pair 8, wherein the sequence of the forward primer is shown in SEQ ID NO:8, and the sequence of the reverse primer is shown in SEQ ID NO:26;

[0017] Primer pair 9, wherein the sequence of the forward primer is as shown in SEQ ID NO:9, and the sequence of the reverse primer is as shown in SEQ ID NO:27;

[0018] Primer pair 10, wherein the sequence of the forward primer is shown in SEQ ID NO: 10, and the sequence of the reverse primer is shown in SEQ ID NO: 28;

[0019] Primer pair 11, wherein the sequence of the forward primer is shown in SEQ ID NO: 11, and the sequence of the reverse primer is shown in SEQ ID NO: 29;

[0020] Primer pair 12, wherein the sequence of the forward primer is shown in SEQ ID NO: 12, and the sequence of the reverse primer is shown in SEQ ID NO: 30;

[0021] Primer pair 13, wherein the sequence of the forward primer is shown in SEQ ID NO: 13, and the sequence of the reverse primer is shown in SEQ ID NO: 31;

[0022] Primer pair 14, wherein the sequence of the forward primer is shown in SEQ ID NO: 14, and the sequence of the reverse primer is shown in SEQ ID NO: 32;

[0023] Primer pair 15, wherein the sequence of the forward primer is shown in SEQ ID NO: 15, and the sequence of the reverse primer is shown in SEQ ID NO: 33;

[0024] Primer pair 16, wherein the sequence of the forward primer is shown in SEQ ID NO: 16, and the sequence of the reverse primer is shown in SEQ ID NO: 34;

[0025] Primer pair 17, wherein the sequence of the forward primer is shown in SEQ ID NO: 17, and the sequence of the reverse primer is shown in SEQ ID NO: 35;

[0026] Primer pair 18, wherein the sequence of the forward primer is shown in SEQ ID NO: 18, and the sequence of the reverse primer is shown in SEQ ID NO: 36;

[0027] or a combination thereof.

[0028] In another preferred embodiment, the forward and reverse primers are designed based on RNA transcribed from positions 1 to 2,900,000 of the Escherichia coli cell genome.

[0029] In another preferred embodiment, the length of the forward primer and the reverse primer is 18-25 nt, preferably 20 nt.

[0030] In another preferred embodiment, the Tm temperature of the forward primer and the reverse primer is 55-60°C, preferably 55°C.

[0031] In another preferred embodiment, the sequence of the forward primer is shown as SEQ ID NO:1, and the sequence of the reverse primer is shown as SEQ ID NO:19.

[0032] The second aspect of the present invention provides a detection reagent, which contains the primer pair described in the first aspect of the present invention.

[0033] In another preferred embodiment, the primer pair is selected from the following group:

[0034] The sequence of the forward primer of the primer pair is shown in SEQ ID NO: 1, and the sequence of the reverse primer of the primer pair is shown in SEQ ID NO: 19;

[0035] The sequence of the forward primer of the primer pair is shown in SEQ ID NO: 2, and the sequence of the reverse primer of the primer pair is shown in SEQ ID NO: 20; or

[0036] The sequence of the forward primer of the primer pair is shown as SEQ ID NO:3, and the sequence of the reverse primer of the primer pair is shown as SEQ ID NO:21.

[0037] In another preferred embodiment, the detection sensitivity of the detection reagent is 0.01-100 pg RNA / μl.

[0038] In another preferred example, the sequence of the forward primer of the primer pair is shown as SEQ ID NO:1, and the sequence of the reverse primer of the primer pair is shown as SEQ ID NO:19.

[0039] The third aspect of the present invention provides a method for detecting whether Escherichia coli RNA exists in a sample to be tested, the method comprising the steps of:

[0040] (S1) providing a sample to be tested, and extracting RNA from the sample to be tested;

[0041] (S2) converting the RNA into cDNA;

[0042] (S3) Using the primer pair described in the first aspect of the present invention, using the cDNA from the sample to be tested as a template, performing PCR, and detecting the PCR amplification product.

[0043] In another preferred embodiment, the sample to be tested is selected from the following group: biorecombinant drug samples, domestic sewage samples and domestic water samples.

[0044] In another preferred embodiment, the sample to be tested contains Escherichia coli RNA.

[0045] In another preferred embodiment, the concentration of E. coli RNA in the sample to be tested is 0.01-10^4 pg / μl

[0046] In another preferred embodiment, the sample to be tested further contains interfering RNA, and the interfering RNA is selected from the following group: probiotic RNA, mammalian cell RNA.

[0047] In another preferred embodiment, the mammalian cell RNA includes cells 293T and cells A549.

[0048] In another preferred embodiment, the detection method is non-diagnostic and non-therapeutic.

[0049] The fourth aspect of the present invention provides a kit, which contains the primer pair described in the first aspect of the present invention.

[0050] In another preferred embodiment, the detection kit also contains a standard control.

[0051] In another preferred embodiment, the standard control is Escherichia coli RNA.

[0052] In another preferred embodiment, the detection kit further comprises a buffer, a reverse transcriptase, a polymerase, or a combination thereof.

[0053] In another preferred embodiment, the kit further comprises instructions.

[0054] In another preferred embodiment, the specification describes the method according to the third aspect of the present invention.

[0055] The fifth aspect of the present invention provides the use of the primer pair as described in the first aspect of the present invention for preparing a detection reagent or a kit for detecting whether Escherichia coli RNA exists in a sample to be tested.

[0056] In another preferred embodiment, the detection reagent or kit is used for product quality control testing.

[0057] In another preferred embodiment, the detection is quantitative detection.

[0058] In another preferred example, the sample to be tested is an environmental sample or a biological product sample.

[0059] In another preferred embodiment, the samples to be tested include biorecombinant drug samples, domestic sewage samples and domestic water samples.

[0060] The sixth aspect of the present invention provides the use of the primer pair as described in the first aspect of the present invention, characterized in that it is used to detect whether Escherichia coli RNA exists in a sample to be tested, thereby monitoring environmental pollution, wherein the sample to be tested is an environmental sample;

[0061] Or it is used to detect whether Escherichia coli RNA exists in the sample to be tested, so as to perform product quality control, wherein the sample to be tested is a biological product sample.

[0062] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The PCR standard curve and melting curve for detecting E. coli RNA using primer pair 1 are shown.

[0064] Figure 2 The PCR standard curve and melting curve for detecting E. coli RNA using primer pair 2 are shown.

[0065] Figure 3 The PCR standard curve and melting curve for detecting E. coli RNA using primer pair 3 are shown. DETAILED DESCRIPTION

[0066] In recent years, DNA sequence analysis has become an ideal tool for bacterial classification, detection and evaluation of phylogenetic relationships. Since conserved sequences such as Escherichia coli rDNA have a high degree of homology within the genus, conserved sequences can be used as target sequences for Escherichia coli detection. After extensive and in-depth research, through a large number of screenings, the inventors have developed a method for rapid detection of Escherichia coli RNA for the first time. Specifically, highly conserved fragments such as 16srRNA are selected in the Escherichia coli genome, and primers are designed and screened based on the RNA sequence of its transcription product. The preferred primer pairs for Escherichia coli RNA obtained by the inventors' screening can not only detect Escherichia coli RNA with high sensitivity, but also distinguish the RNA of Escherichia coli from the RNA of other eukaryotic cells (such as probiotics, 293T cells, A549 cells, etc.), thereby obtaining a detection method for detecting Escherichia coli RNA with both specificity and sensitivity. The method of the present invention has the characteristics of wide application range, simple and fast operation, high specificity and high sensitivity. The present invention has been completed on this basis.

[0067] the term

[0068] As used herein, the term "E. coli RNA" refers to an RNA molecule formed by transcription based on the DNA sequence of the E. coli genome. E. coli RNA is the main source of host residual RNA in recombinant biological products using E. coli as an engineered bacterium.

[0069] Primers and primer design

[0070] The term "primer" as used herein has the meaning commonly understood by those skilled in the art. At the initiation of nucleotide polymerization, the primer is complementary to a cDNA template strand at one end of the target gene to guide the synthesis of a new nucleic acid strand.

[0071] The primers for PCR reactions are generally designed and synthesized artificially. The purpose of PCR primer design is to find a pair of suitable nucleotide fragments that can effectively amplify the template DNA sequence. The quality of the primers is directly related to the specificity and success of PCR.

[0072] There are three basic principles to follow in primer design: first, the sequences of the primer and template should be closely complementary; second, the formation of stable dimers or hairpin structures should be avoided between primers; and third, the primer should not induce DNA polymerization at non-target sites on the template (i.e., mismatch).

[0073] Although the above principles of primer design are known, in actual application, the samples to be tested may contain a large number of various nucleic acid substances, including DNA, RNA and other nucleic acid substances from different organisms or the same organism, which may affect the specificity and sensitivity of PCR.

[0074] In addition, differences in factors such as the PCR system will also lead to changes in the specificity and sensitivity of PCR. For example, some representative influencing factors include (but are not limited to): many factors, such as primer length, product length, sequence Tm value (melting temperature), internal stability (internal stability, reflected by ΔG value) of double-stranded formation between primers and templates, energy value of forming primer dimers and duplex formation and hairpin, priming efficiency at false priming sites, GC content (composition) of primers and products, etc. Therefore, it is still a great challenge to obtain primers with high sensitivity and high specificity.

[0075] In the present invention, the inventors designed dozens of pairs of primers based on the primer design principle, and selected 18 pairs of preferred primers. Through screening and verification, the results showed that 3 pairs of primers had excellent comprehensive performance, extremely high specificity and extremely high sensitivity.

[0076] Preparation of standards

[0077] Standards are an important part of the qPCR detection method. To quantify the target gene in the sample to be tested, it is first necessary to establish a linear relationship between the Ct value and the copy number / concentration, that is, the standard curve. The standard curve needs to be generated by the standard. The gene sequence in the standard must be consistent with the target gene sequence in the sample to be tested, so as to ensure that the primer amplification efficiency in both is exactly the same. The source of the standard can be a plasmid, a synthetic and purified PCR product, a purified RNA reversed cDNA or genomic DNA, etc. The standard curve is generated by different gradient standards, and each standard gradient establishes a relationship between the Ct value and the logarithmic value of the copy number / concentration. The standard and the sample to be tested react at the same time. After substituting the Ct value of the sample to be tested into the standard curve, the copy number / concentration of the target gene in the sample to be tested can be converted.

[0078] Standards can be prepared by self-extraction using reagents, or prepared and synthesized using commercial RNA extraction kits.

[0079] Detection Reagents

[0080] The present invention also provides a detection reagent for detecting Escherichia coli cell genome RNA, wherein the detection reagent comprises the primer pair of the present invention.

[0081] Detection Kits

[0082] The present invention also provides an Escherichia coli RNA detection kit, which comprises the primer pair, standard substance, buffer, enzyme and the like of the present invention.

[0083] In a specific embodiment, the kit of the present invention also contains other components required for carrying out the detection and instructions for use of the kit for detection. In a preferred embodiment, the kit also contains a standard control.

[0084] Detection Methods

[0085] Based on the primer pair or detection reagent of the present invention, the present invention further provides a method for detecting E. coli cell RNA, the method comprising: using the primer pair or detection reagent of the present invention, performing PCR on the sample to be tested, and detecting the PCR amplification product. In a specific embodiment, the method is used to determine whether the food or health product is produced from E. coli.

[0086] The main advantages of the present invention include:

[0087] (a) This method has high detection sensitivity and the detection limit can reach 0.01pg / μl.

[0088] (b) The present invention can effectively distinguish E. coli RNA from RNA of other types of sources, such as RNA from probiotics, eukaryotic cells 293T, and eukaryotic cells A549, with high specificity;

[0089] (c) The method of the present invention has a wide range of applications, is simple to operate and has low cost.

[0090] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0091] Example 1 Primers and Primer Design

[0092] Based on the genome of E. coli, the present invention designs and optimizes 18 pairs of primer sequences for amplifying E. coli RNA as shown in Table 1. The common characteristics of these preferred primer pairs are: length of 18-25 nt, Tm of 55-60°C, GC content of 40%-65%:

[0093] Table 1

[0094]

[0095] Example 2 RNA sample preparation method

[0096] 20ul of activated JM109 Escherichia coli (purchased from Takara) was extracted, and the total RNA of the Escherichia coli host was extracted using the Trizol method, and then dissolved in 10ul DEPC water.

[0097] The specific steps are as follows: add 1 ml of Trizol to 20 ul of activated Escherichia coli solution, then add chloroform at a ratio of 1:0.2, then add isopropanol at a ratio of 1:0.5, add separation solution at a ratio of 1:1 (phenol: chloroform: isoamyl alcohol = 25:24:1), then precipitate with isopropanol and wash with 75% ethanol.

[0098] The purified RNA samples were tested by micro-ultraviolet spectrophotometry. The test results are shown in Table 2.

[0099] Table 2

[0100] Sample ID Sample type Concentration (ug / ul) 260 / 280 E.coli (20ul) RNA 589.2 1.88 E.coli (20ul) RNA 586.4 1.87 E.coli (20ul) RNA 580.0 1.87 water RNA 0.4 -1

[0101] The results showed that the purity of RNA samples prepared by phenol-chloroform purification met the standard.

[0102] Example 3 Primer Pair Amplification Effect

[0103] This example demonstrates the excellent amplification effect by establishing a standard curve for primer pairs 1, 2, and 3:

[0104] 3.1 Preparation of E. coli RNA standards

[0105] The concentration of total RNA extracted from pure culture of E. coli was adjusted to prepare the standard.

[0106] Take out JM109 Escherichia coli strain (Takara, Cat. 9052) from the -80°C freezer, thaw at room temperature, and add to 50 ml of LB medium prepared in advance. Incubate at 37°C, 200 rpm overnight.

[0107] 1 ml of activated E. coli solution was taken and total RNA was extracted using Qiagen Total RNA Extraction Kit 50T. RNA was re-dissolved in DEPC water. The concentration and purity were measured using a micro-UV spectrophotometer and stored at -80°C after aliquoting.

[0108] 3.2 PCR amplification

[0109] The RNA sample was placed on ice to melt and mix before reverse transcription, and the reverse transcription reaction was performed according to the reagent instructions. The reaction system was: AMV reverse transcriptase (BBI, 10U / μl) 0.5μl, AMV RT buffer (5X) 2μl, Oligo dT primer (Takara, 50μM) 0.5μl, random primer (Takara, 100μM) 0.5μl, dNTP Mixture (Takara, 10mM) 1μl, RNA (4ug~8ug) 4μl, DEPC water 1.5μl.

[0110] To the cDNA generated by reverse transcription, 1 μl of upstream primer (10 μM) and 1 μl of downstream primer (10 μM) were added, and the system was configured according to the reagent instructions and qPCR was started.

[0111] 3.3 Amplification effect of primer pair 1

[0112] The preparation method of E. coli RNA standard is the same as 3.1. Adjust the starting concentration of E. coli RNA standard to 10^3pg / μl, dilute 10-fold to 6 points, and prepare 10^3-0.01pg / μl standard. The PCR amplification experimental method is the same as 3.2.

[0113] The experimental results are shown in Table 3 and Figure 1 As shown: The amplification efficiency of primer pair 1 reached 86.37%, which met the requirements of qPCR quantitative detection. In the concentration range of 0.01pg / μl to 1,000pg / μl, the linearity was good and the amplification was stable. The detection limit of this method can reach 0.01pg / μl, and the linear correlation coefficient R of the standard curve is 0.997, which has a high correlation.

[0114] Table 3 Primer pair 1 amplification standard curve data

[0115]

[0116] 3.4 Amplification effect of primer pair 2

[0117] The preparation method of E. coli RNA standard is the same as 3.1. Adjust the starting concentration of E. coli RNA standard to 10^4pg / μl, dilute 5 points tenfold, and prepare 10^4pg / μl~1pg / μl standard. The PCR amplification experimental method is the same as 3.2.

[0118] The experimental results are shown in Table 4 and Figure 2 As shown: The amplification efficiency of primer pair 2 reached 95.36%, which met the requirements of qPCR quantitative detection. In the concentration range of 1pg / μl to 10,000pg / μl, the linearity was good and the amplification was stable. The detection limit of this method was 1pg / μl, and the linear correlation coefficient R of the standard curve was 0.999, with high correlation.

[0119] Table 4. Primer pair 2 amplification standard curve data

[0120]

[0121] 3.5 Amplification effect of primer pair 3

[0122] The preparation method of E. coli RNA standard is the same as 3.1. Adjust the starting concentration of E. coli RNA standard to 10^3pg / μl, dilute it tenfold to 6 points, and prepare 10^3pg / μl~0.01pg / μl standard. The PCR amplification experimental method is the same as 3.2.

[0123] The experimental results are shown in Table 5 and Figure 3 As shown: The amplification efficiency of primer pair 3 reached 96.01%, which met the requirements of qPCR quantitative detection. In the concentration range of 0.01pg / μl to 1,000pg / μl, the linearity was good and the amplification was stable. The detection limit of this method reached 0.01pg / μl, and the linear correlation coefficient R of the standard curve was 0.99, with high correlation.

[0124] Table 5. Primer pair 3 amplification standard curve data

[0125]

[0126] In this example, the amplification effects of primer pairs 4-18 in Example 1 were also verified, and all met the requirements for qPCR quantitative detection, with good linearity and stable amplification within the concentration range of 0.01 pg / μl to 1,0000 pg / μl.

[0127] Example 4: Detection of E. coli host RNA residues in recombinant biological products using primer pair 1, and differentiation of E. coli and probiotic RNA

[0128] In this example, primer pair 1 was used to detect host RNA residues in three recombinant biological product drug samples with Escherichia coli as the engineering bacteria to investigate the feasibility of the method. At the same time, RNA extracted from probiotic culture was added to the samples to investigate the recovery rate to confirm the specificity of the method. The SD value of the sample detection was calculated to represent the precision of the method.

[0129] The preparation of E. coli RNA standard was the same as in Example 3.

[0130] Prepare specific test samples: Take the E. coli RNA standard and prepare 0.5, 5, and 50 pg / μl concentrations of each sample spiked with 10 ng of RNA extracted from the probiotic culture.

[0131] The PCR amplification method is the same as in Example 3.

[0132] result

[0133] The test results are shown in Table 6. In the biological drug samples tested by this method, the E. coli RNA content was lower than 1 pg / μl, and the E. coli RNA and probiotic RNA could be clearly distinguished.

[0134] Table 6

[0135]

[0136]

[0137] Example 5: Detection of E. coli RNA in domestic sewage samples using primer pair 2, and differentiation of certain medicinal E. coli and eukaryotic cell 293T RNA

[0138] In this example, primer pair 2 was used to detect Escherichia coli RNA in three domestic sewage samples to investigate the feasibility of the method. RNA extracted from eukaryotic cell 293T culture was added to the samples to investigate the recovery rate to confirm the specificity of the method. The SD value of the sample detection was calculated to represent the precision of the method.

[0139] The preparation method of Escherichia coli RNA standard is the same as that in Example 3.

[0140] Prepare specific detection samples: Take E. coli RNA standard and prepare 5, 50, 500, and 5000 pg / μl concentrations, and add 10 ng of each sample of RNA extracted from 293T cell culture.

[0141] The PCR amplification method is the same as in Example 3.

[0142] Experimental Results

[0143] As shown in Table 7, the E. coli RNA content in the domestic sewage samples detected by this method was lower than 10 pg / μl, and the E. coli RNA and eukaryotic cell 293T RNA could be clearly distinguished.

[0144] Table 7

[0145]

[0146]

[0147] Example 6: Detection of E. coli RNA in domestic water samples using primer pair 3, and differentiation of a certain medicinal E. coli from eukaryotic cell A549 RNA

[0148] In this example, primer pair 3 was used to detect Escherichia coli RNA in three domestic water samples to investigate the method feasibility. RNA extracted from eukaryotic cell A549 culture was added to the samples to investigate the recovery rate to confirm the method specificity. The SD value of sample detection was calculated to represent the method precision.

[0149] The preparation method of E. coli RNA standard is the same as that in Example 3.

[0150] Prepare specific detection samples: Take E. coli RNA standard and prepare 0.5 and 5 pg / μl concentrations, and add 10 ng of RNA extracted from A549 cell culture to each sample.

[0151] The PCR amplification method is the same as in Example 3.

[0152] result

[0153] As shown in Table 8, the E. coli RNA content in the domestic water samples tested by this method can be lower than 1 pg / μl, and the E. coli RNA and the eukaryotic cell A549 RNA can be clearly distinguished.

[0154] Table 8

[0155]

[0156] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A primer pair for detecting Escherichia coli RNA, characterized in that: The primer pair comprises a forward primer and a reverse primer, and the primer pair is selected from the group consisting of: (a) Primer pair 1, wherein the sequence of the forward primer is shown in SEQ ID NO: 1, and the sequence of the reverse primer is shown in SEQ ID NO: 19; (b) Primer pair 2, wherein the sequence of the forward primer is shown in SEQ ID NO: 2, and the sequence of the reverse primer is shown in SEQ ID NO: 20; (c) Primer pair 3, wherein the sequence of the forward primer is shown in SEQ ID NO: 3, and the sequence of the reverse primer is shown in SEQ ID NO: 21; or a combination thereof.

2. A detection reagent, characterized in that: The detection reagent contains the primer pair according to claim 1.

3. A method for detecting whether Escherichia coli RNA exists in a sample to be tested, characterized in that: The method comprises the steps of: (S1) providing a sample to be tested, and extracting RNA from the sample to be tested; (S2) converting the RNA into cDNA; (S3) Using the primer pair described in claim 1, using the cDNA from the sample to be tested as a template, performing PCR, and detecting the PCR amplification product.

4. A kit, characterized in that: The kit contains the primer pair according to claim 1.

5. The use of the primer pair according to claim 1, characterized in that: Used to prepare a detection reagent or kit for detecting whether Escherichia coli RNA exists in a sample to be tested.

6. The use according to claim 5, characterized in that The detection reagent or kit is used for product quality control detection.

7. The use according to claim 5, characterized in that The detection is a quantitative detection.

8. The use according to claim 5, characterized in that The sample to be tested is an environmental sample or a biological product sample.

9. The use according to claim 5, characterized in that The samples to be tested include biorecombinant drug samples, domestic sewage samples and domestic water samples.

10. The use of the primer pair according to claim 1, characterized in that: Used to detect whether Escherichia coli RNA exists in the sample to be tested, so as to monitor environmental pollution, wherein the sample to be tested is an environmental sample; Or it is used to detect whether Escherichia coli RNA exists in the sample to be tested, so as to perform product quality control, wherein the sample to be tested is a biological product sample.

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