LU503460B1

LU503460B1 - PCR amplification primer pair and probe, and applications thereof

Info

Publication number: LU503460B1
Application number: LU503460A
Authority: LU
Inventors: Qiang Wang; Qingping Zhang; Feng Zhang; Teng Qi; Xu Zhu; Jing Wang
Original assignee: Chongqing Tianyou Dairy Co Ltd
Priority date: 2022-09-26
Filing date: 2023-02-09
Publication date: 2023-08-10
Also published as: CN115838812A

Abstract

PCR amplification primer pair and probe, and applications thereof Present invention belongs to the technical field of microorganism detection, and specifically, relates to a PCR amplification primer pair and a probe, and applications thereof. An forward primer of the PCR amplification primer pair includes a sequence shown as SEQIDNO:1, and a reverse primer includes a sequence shown as SEQIDNO:2; and the probe includes a sequence shown as SEQIDNO:3. The PCR amplification primer pair and the probe are highly specific to Bacillussporothermodurans (BSTD), and may start a curve at about 20 cycles (about 1h), such that detection time is short; and the limit of detection may reach 0.75 CFU/mL, such that high sensitivity may be achieved. In addition, under the action of the probe, PCR amplification may be monitored in real time during PCR. Bacterium enrichment is not required in the first stage, and gel electrophoresis is not required subsequently, such that operation steps are simplified, thereby further facilitating detection.

Description

PCR amplification primer pair and probe, and applications thereof LU503460

Technical Field

The present invention belongs to the technical field of microorganism detection, and specifically, relates to a PCR amplification primer pair and a probe, and applications thereof.

Background

Bacillus is a kind of bacilli or cocci that can form spores, which is an aerobic or partly anaerobic gram-positive bacteria. The formed spores are highly resistant to high temperatures, high permeability, acids and bases. The Bacillus is widely distributed in nature (for example, air, soil and plants), and may enter food by means of various ways, such as soil, dust or a biomembrane. Therefore, the bacteria (including bacillus amyloliquefaciens, bacillus licheniformis, bacillus subtilis and bacillus cereus) are the most common microorganisms in food and food raw materials, especially in bean products such as thick broad-bean sauce, fermented bean curd and soy sauce, dairy products, and fruit and vegetable fermented food such as pickled vegetables and ketchup. The bacteria are not only numerous in number, but also high in detection rate.

Bacillus pasteurization is a sterilization mode commonly used in food enterprises.

However, pasteurization is difficult to completely kill spores, especially the spores of the

BSTD (which is the Bacillus that can withstand pasteurization temperatures). The heat resistance of the bacillus varies with different types of bacteria. Some of the bacillus can even withstand high temperatures of 100°C and above. The presence of the BSTD in food may bring certain influence to the quality of the food. Some BSTD can produce toxins and be pathogenic, and the ubiquity of the BSTD poses a huge challenge to the food industry. The

BSTD is typical, and is a kind of bacteria that have relatively-high heat resistance and produce endospore. The bacteria belong to bacteria, Firmicutes, bacilli, Bacillales, Bacillaceae,

Genus Bacillus, BSTD species. The model strain number of the strain is M215 (DSMZ 10599).

In 1985 and 1990, the BSTD is discovered in Ultraheattreated (UHT) milk in Germany.

Subsequent discoveries have been made in other European countries (such as, Belgium,

France and Spain). In some foods or environments, these BSTD withstanding high temperatures can survive and reproduce without significantly altering the quality of the cow’s milk or being pathogenic, such that the BSTD is easily overlooked. Although there are no studies to show that the BSTD is not pathogenic and does not cause significant quality reduction in products, the BSTD is widely present in the environments and foods. The detection of the BSTD may laterally reflect the presence of defects in a production proceds503460 which may bring potential risks to food safety and food quality control. For example, in a production process of the dairy products, the national standard GB-4789.2 requires that the total plate count in the milk cannot exceed 10° CFU/ml. If the BSTD is not thoroughly sterilized after activation in the early stage, the bacteria will contaminate the whole production line of the dairy products, causing the total plate count to exceed the standard, and also laterally reflecting that there are certain loopholes in the production process. Therefore, enterprises, especially the food industry, are required to have comprehensive and in-depth understanding of the BSTD, so as to establish a more perfect microorganism control system.

So far, traditional methods for detecting the BSTD include a plate culture method, a 16srDNA identification method or a dye method, and there is no systematic method specifically for detecting the BSTD in China. The traditional plate culture method needs to culture the bacteria for about 48h-72h, and then perform observation with naked eyes.

However, the colony morphology of the bacteria on the plate is extremely inconspicuous, such that the bacteria are easily overlooked, thereby affecting a detection result. The 16srDNA identification method usually uses bacterial universal primers 27F and 1492R. However, the process needs to perform bacteria enrichment in the early stage, and bacteria enrichment needs about 24h-48h. In addition, the PCR identification method further needs to perform gel electrophoresis, such that operation steps are relatively tedious, primer specificity is weak, and a sequencing result may show a false positive result. The dye method does not specifically identify a PCR product. Assuming that the PCR product contains non-target product DNA, which is “undesired DNA”. However, the DNA may not be recognized by a dye, as long as it is a double-stranded DNA, the dye may be combined with the DNA and emit light. In this way, it is difficult to determine whether detected fluorescence is from the “desired DNA” or the “undesired DNA”. To sum up, the current commonly-used detection methods have disadvantages of weak specificity, low sensitivity, long time consumption and complicated operation steps.

Therefore, A BSTD detection method with high specificity, high sensitivity, short time consumption and concise operation steps is necessary.

Summary

In view of the above problems, one of the objectives in the present invention is to provide a PCR amplification primer pair and a probe. The PCR amplification primer pair and the probe may specifically identify the BSTD, and are highly specific; a curve may be started at about 20 cycles, such that short time consumption is achieved; and the limit of detectidd)503460 may reach 0.75 CFU/mL, such that high sensitivity may be achieved. In addition, under the action of the probe, PCR amplification may be monitored in real time during PCR, thereby further facilitating detection.

In order to achieve the above purpose, the present invention may use the following technical solutions.

An aspect of the present invention provides a PCR amplification primer pair and a probe.

An forward primer includes a sequence shown as SEQIDNO:1, and a reverse primer includes a sequence shown as SEQIDNO:2; and the probe includes a sequence shown as SEQIDNO:3.

Another aspect of the present invention provides a detection reagent. The detection reagent may include the PCR amplification primer pair and the probe.

Still another aspect of the present invention provides a detection kit. The detection kit may include the PCR amplification primer pair and the probe or the detection reagent.

Still another aspect of the present invention provides applications of the PCR amplification primer pair and the probe or the detection reagent or the detection kit in detection of BSTD.

Still another aspect of the present invention provides a PCR amplification detection method for Bacillussporothermodurans. The method includes: performing PCR amplification on a sample to be detected by using the PCR amplification primer pair and the probe, and during PCR amplification, according to a fluorescence signal, determining whether the sample to be detected contains BSTD; and according to a CT value and a standard curve, quantitatively analyzing the BSTD of the sample to be detected.

The present invention has the following beneficial effects. The PCR amplification primer pair and the probe provided in the present invention are highly specific to the BSTD, and may start a curve at about 20 cycles (about 1h), such that detection time is short; and the limit of detection may reach 0.75 CFU/mL, such that high sensitivity may be achieved. In addition, under the action of the probe, PCR amplification may be monitored in real time during PCR.

Bacterium enrichment is not required in the early stage, and gel electrophoresis is not required subsequently, such that operation steps are simplified, thereby further facilitating detection.

Brief Description of the Drawings

Fig. 1 is a microscopic examination chart of BSTD B1 after activation.

Fig. 2 shows a phylogenetic tree of BSTD BI.

Fig. 3 shows initial establishment of a fluorescent quantitative PCR method.

Fig. 4 shows the impact of different annealing temperatures on detection of BSTD B1. LU503460

Fig. 5 shows specificity evaluation of a fluorescent quantitative PCR method.

Fig. 6 shows sensitivity evaluation of a fluorescent quantitative PCR method.

Fig. 7 shows a detection result of an artificially-contaminated sample

Fig. 8 shows detection results of different dairy products available in the market.

Detailed Description of the Embodiments

The embodiments are given to better describe the present invention, but the content of the present invention is not limited only to the given embodiments. Therefore, non-essential improvements and adjustments to the embodiments made by a person skilled in the art in accordance with the content of the above present invention still fall within the scope of protection of the present invention.

The terms used herein are only intended to describe specific embodiments and are not intended to limit the present invention. Expressions in the singular form include those in the plural form unless the expressions have a distinctly different meaning in the context. As used herein, it is to be understood that terms such as "include", "have", "contain", and the like are intended to indicate the presence of features, figures, operations, components, parts, elements, materials, or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, figures, operations, components, parts, elements, materials, or combinations thereof may exist or may be added. As used here, "/" may be interpreted as "and" or "or", as appropriate.

An embodiment of the present invention provides a PCR amplification primer pair and a probe. An forward primer includes a sequence shown as SEQIDNO:1, and a reverse primer includes a sequence shown as SEQIDNO:2; and the probe includes a sequence shown as

SEQIDNO:3.

It is to be noted that, in the present invention, by means of performing 16srDNA sequencing on BSTD, a sequence is compared with 16srDNA of a standard mode strain M215, and 100% homology is reached; according to the 16srDNA sequence of the BSTD and a sequence of related Bacillus, comparison is performed by using DNAMAN, so as to find a highly conserved region of the strain and determine V1-V9 variable regions, and specific primers are designed in the variable regions by means of PrimerPremier6 and Oligo6; and thus, the PCR amplification primer pair shown as SEQIDNO:1 and SEQIDNO:2 and the probe shown as SEQIDNO:3 in the present invention are obtained; the primer pair and the probe are highly specific to the BSTD, and may start the curve at about 20 cycles (about 1h),

such that detection time is short; and the limit of detection may reach 0.75 CFU/mL. In son&J503460 specific embodiments, 7-8 BSTD may be detected from 10 mL of the sample to be detected, indicating that the sensitivity is high. In addition, after the probe is added, a synthesized double strand may be labeled during PCR amplification to form the double strand. By means 5 of some external devices such as a PCR fluorescence detector, changes in a fluorescence signal may be monitored, such that PCR amplification may be detected in real time, thereby further facilitating detection. In addition, in the present invention, the probe is designed.

Compared with a conventional PCR amplification detection method, in the present invention, gel electrophoresis is not required to be performed on the PCR amplification product. Due to high sensitivity, bacterium enrichment is not required in the early stage, such that the operation steps may be more concise, and the detection time may be shorter.

In addition, it is to be understood that, for enterprises, the shortening of the time for detecting the BSTD may reduce the risk level of microorganism quality control to a great extent; and monitoring of all aspects of environments, raw materials and devices may be established, thereby building an impenetrable barrier to enterprise production for the BSTD.

In some specific embodiments, in the PCR amplification primer pair and the probe, the probe is a Tagman probe. It is to be noted that, for different types of probes (for example, a

FRET hybridization probe, a Hybridization probe or a Molecular Beacon), sequences of the designed probes are different. The sequence of the probe in the present invention is suitable for the Tagman probe.

In some specific embodiments, in the PCR amplification primer pair and the probe, a 3’ terminal of the probe is connected to a fluorescence quenching group, and a 5’ terminal is connected to a fluorescence reporter group. It is to be noted that, a TaqMan method qPCR is a method of using a TaqMan fluorescence probe for fluorescence detection. The basic principle of the method is to add, during PCR amplification, a specific fluorescence probe while a pair of primers is added. Two terminals of the probe are respectively labeled as the fluorescence reporter group and the fluorescence quenching group. At the beginning, the probe is completely bound to any single strand of DNA, a fluorescence signal emitted by a reporter group is absorbed by a quenching group, and the fluorescence signal is not detected; during

PCR amplification, the 5'-3' nucleic acid exonuclease activity of a Taq enzyme cleaves and degrades the probe, to cause the fluorescence reporter group to separate from the fluorescence quenching group, so as to emit light; and the number of the cleaved fluorescent molecules is proportional to the number of PCR products. Therefore, the purpose of detecting the amplification amount of the PCR products may be achieved by means of detecting the fluorescence intensity of a PCR reaction system. LU503460

In some specific embodiments, in the PCR amplification primer pair and the probe, the fluorescence quenching group is MGB, and the fluorescence reporter group is FAM. It is to be noted that, according to different detection purposes (for example, two different detection purposes of detecting strains and detecting proteins), different groups are required to be selected. In addition, for the same detection purpose, for example, strain detection, different groups also have different results for different target bacteria. Therefore, it is very important to select the right group. In the present invention, preferably, the fluorescence quenching group is MGB, and the fluorescence reporter group is FAM.

Another embodiment of the present invention provides a detection reagent. The detection reagent includes the PCR amplification primer pair and the probe. It is to be noted that, the

PCR amplification primer pair and the probe may be combined with a conventional auxiliary reagent to form a detection reagent used for detecting the BSTD. The conventional auxiliary reagent is known in the art, for example, a PCR reaction buffer solution in a PCR amplification reaction system, DNA polymerase, or dNTP On the basis of the PCR amplification primer pair and the probe in the detection reagent, the detection reagent in the embodiments of the present invention can also implement qualitative and quantitative analysis on the BSTD, and has the advantages of being high in specificity, short in time consumption and high in sensitivity.

Still another embodiment of the present invention provides a detection kit. The detection kit includes the PCR amplification primer pair and the probe or the detection reagent. It is to be noted that, the composition of the detection kit is in a conventional form known in the art.

For example, the detection kit is provided with a kit specification. For example, there are reagent bottles for holding various detection reagents. For example, several small lattices are arranged to place detection reagent bottles. For example, a pipette is used to pipette the detection reagent. Likewise, on the basis of the PCR amplification primer pair and the probe in the detection kit, the detection kit in the embodiments of the present invention can also implement qualitative and quantitative analysis on the BSTD, and has the advantages of being high in specificity, short in time consumption and high in sensitivity.

Still another embodiment of the present invention provides applications of the PCR amplification primer pair and the probe or the detection reagent or the detection kit in detection of BSTD. It is to be noted that, when the PCR amplification primer pair and the probe or the detection reagent is used to amplify the BSTD, strong fluorescent light may be produced during amplification, facilitating detection and analysis. In addition, the PCR amplification primer pair and the probe or the detection reagent may also be combined withl4)503460 microfluidic chip to apply to the detection of the BSTD. In combination with the high efficiency and convenience of the microfluidic chip, great convenience may be brought for the detection of the BSTD.

Still another embodiment of the present invention provides a PCR amplification detection method for Bacillussporothermodurans. The method includes: performing PCR amplification on a sample to be detected by using the PCR amplification primer pair and the probe, and during PCR amplification, according to a fluorescence signal, determining whether the sample to be detected contains BSTD; and according to a CT value and a standard curve, quantitatively analyzing the BSTD of the sample to be detected.

In some specific embodiments, in the PCR amplification detection method for

Bacillussporothermodurans, an annealing temperature of a PCR amplification reaction may range from 55°C to 65°C. It is to be noted that, the annealing temperature mainly depends on the base composition, length and concentration of a primer; and an appropriate annealing temperature is particularly critical for the specificity of the PCR amplification reaction.

Generally, the annealing temperature affects both the specificity and sensitivity (amplification efficiency) of the PCR reaction, and the specificity and the sensitivity are negatively correlated. That is to say, the low annealing temperature may improve the sensitivity of the

PCR reaction, but the specificity is relatively poor; and the high annealing temperature may improve the specificity of the reaction but reduce the amplification efficiency. The annealing temperature in the present invention is set to 55C-65"C, for example, 56°C, 58°C, 60°C, 62°C and 64°C, such that the PCR amplification primer pair and the probe may maintain high specificity in the PCR amplification reaction, and the rate of the PCR amplification reaction may also be maintained at a high level.

In some specific embodiments, in the PCR amplification detection method for

Bacillussporothermodurans, the sample to be detected is milk. It is to be noted that, the sample to be detected may be an extracted microorganism DNA template, or may directly be the milk. Taking the primer pair and the probe and convenience into consideration, the milk may be directly detected. Even if the milk contains some substances inhibiting the PCR reaction, the primer pair and the probe in the present invention directly detect the milk without affecting the accuracy of detection results. In addition, the detection method can not only distinguish other common foodborne pathogenic bacteria from some common bacteria, but also well discriminate related bacillus cereus and bacillus subtilis.

In order to better understand the present invention, the content of the present invention is further described below with reference to specific embodiments, but is not only limited to thé/503460 following examples.

In the following embodiments, listeria monocytogenes ATCC19115, escherichia coli

ATCC8739, bacillus cereus CMCC(B)63303, bacillus subtilis ATCC6633 and citrobacter freundii ATCC43864 are all purchased from the China Center of Industrial Culture Collection (CICC); Bacillussporothermodurans M215 (DSMZ10599) is purchased from Deutsche

Sammlung von Mikroorganismen und Zellkulturen (DSMZ); and the BSTD B1 is from milk.

In the following embodiments, a fluorescent quantitative PCR reaction system is shown in Table 1 below.

Table 1 Fluorescent quantitative PCR reaction system amo vo

Embodiment 1 Separation, screening and identification of BSTD B1 (1) Preliminary screening of the BSTD: a sample is first heated for 30 min at 100°C and then poured into a BHI medium, the medium is cultured for 48-72h at 55°C, single colonies are then selected, and purified and cultured strains are stored in a -80°C freezer. (2) Activation and identification of the strains: a glycerin tube is taken and inoculated in the sterilized liquid BHI medium, cultured for 24-48h at 37°C, continuously activated for 3 times until no bacteria are found by microscopic examination, and then inoculated into the

BHI medium with 2% inoculation amount; and bacterial fluid is taken and sent to Sangon

Biotech (Shanghai) Co., Ltd. for sequencing. (3) Separation and identification of the BSTD: Blast is performed in NCBI after the

BSTD separated and identified from the sample is sequenced, the strain is found to be highly homologous to a type strain BSTD M215, and the homology a 16srDNA sequence reaches 100%; a phylogenetic tree is shown in Fig. 2, indicating that the strain belongs to Bacillus sporothermodurans; and the strain is named as the BSTD B1, which is shown in Fig. 1.

Embodiment 2 Design of a fluorescent quantitative PCR primer (1) Activation of strains

Bacillus cereus, bacillus subtilis, listeria monocytogenes, escherichia coli and citrobacter freundii are inoculated into an LB broth medium, and are cultured at 37°C a logarithmk&/503460 phase for later use; and the BSTD B1 and the BSTD M215 are inoculated into a BHI medium, and are cultured at 37°C a logarithmic phase for later use. (2) Design of a primer and a probe

According to the 16srDNA sequence of the BSTD B1 and a sequence of related Bacillus, comparison is performed by using DNAMAN, so as to find a highly conserved region of the strain and determine V1-V9 variable regions; specific primers and probes are designed in the variable regions by means of BeaconDesigner and Oligo6; a 6-FAM fluorescence group is labeled on a 5’ terminal of a fluorescence probe, and a quenching group MGB which can quench a fluorescence signal emitted by the fluorescence group is labeled on a 3° terminal; and by means of calculating a Tm value and verifying the specificity of the primer in NCBI, the sequences of the designed primer and probe are finally sent to Sangon Biotech (Shanghai)

Co., Ltd. for synthesis.

Primer sequence:

B1-F forward primer sequence (SEQIDNO:1): GAGTGCAGAAGAGAAGAG.

B1-R reverse primer sequence (SEQIDNO:2): GTCAGTTACAGACCAAAGA.

Fluorescence probe sequence (SEQIDNO:3): 5’-6-FAM-TTCGCCACTGGTGTTCCTCC-MGB-3’.

Embodiment 3 Establishment of a fluorescent quantitative PCR detection method of

BSTD

(1) DNA preparation of positive control BSTD M215

DNA extraction is performed on the positive control BSTD M215 in Embodiment 2 cultured to the logarithmic phase by using an Ezup column bacterial genome DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd.); the extracted DNA is preserved at -80°C for later use as a PCR reaction positive control template, a NanoDropOne ultramicro ultraviolet spectrophotometer is used to measure a DNA concentration. (2) DNA preparation of BSTD B1 to be detected

In order to use a high-temperature pyrolysis method to extract DNA of the BSTD B1: 1 mL of bacterial fluid of the BSTD B1 cultured to the logarithmic phase is taken and placed into a 1.5 mL sterile Eppendorf centrifuge tube, and centrifugation is performed for 5 min at 8000 rpm at 4°C; supernatant is sucked and added to another 1.5 mL sterile Eppendorf centrifuge tube, centrifugation is performed for 10 min at 8000 rpm, and then the supernatant is discarded; 200 uL of deionized water is added to resuspend the bacteria, heated for 10 min in a boiling water bath, then immediately placed in liquid nitrogen and taken out after cooling;

then centrifugation is performed for 10 min at 8000 rpm, and the supernatant is taken asL4J503460

PCR reaction template. (3) Counting of BSTD M215 and BSTD B1

After the BSTD M215 and the BSTD B1 which are cultured to the logarithmic phase are diluted to different gradients, plate spreading and counting is performed; and there are 3 repeats in each gradient, and a spread plate is inverted in a 37°C constant temperature incubator and cultured for about 48h. (4) Establishment of a fluorescent quantitative PCR method

The extracted DNA of the BSTD M215 is used as a positive control template; the DNA of the BSTD B1 extracted by means of the high-temperature pyrolysis method is used as a template of the sample to be detected; ddH»O is used as a negative control. After the positive control BSTD M215 is extracted by using an Ezup column bacterial genome DNA extraction kit, the DNA concentration of the bacteria is 135.4 ng/ul, and the DNA extracted from the bacteria is used as a template standard and stored at -80°C for later use. By means of plate spreading and counting, it is learned that the viable counts of the BSTD M215 and the BSTD

B1 cultured to the logarithmic phase are respectively 1.8x10°CFU/ml and 2.0x10°CFU/ml. A q-PCR reaction system is shown in Table 1 above, and a PCR reaction procedure is shown in

Table 2 below. Then q-PCR reaction detection is performed.

Table 2 PCR reaction procedure in Embodiment 3

CO

After the PCR reaction is completed, an amplification curve is analyzed in visual fluorescence quantitative analysis software, and the Ct value in a standard curve is used to determine whether the BSTD is detected. Fluorescence quantitative PCR is shown in Fig. 3.

Both the positive control BSTD M215 and a strain B1 to be detected started start a curve at about 16 Ct values, and a negative control do not start a curve, indicating that an amplification effect is good.

Embodiment 4 Impact of different annealing temperatures on detection of BSTD B1

A series of control reactions are set to determine the optimal annealing temperature.

From 55°C to 65°C, 8 temperature gradients are set. The 8 temperature gradients are respectively 55.0°C, 55.7°C, 57.0°C, 59.0C, 61.4°C, 63.3°C, 64.5°C and 65°C, and a contrblJ503460 is ddH>O. The q-PCR reaction system is shown in Table 1 above, and the PCR reaction procedure is shown in Table 3 below. Then q-PCR detection is performed separately.

Table 3 PCR reaction procedure in Embodiment 4 wo

A q-PCR detection result is shown in Fig. 4. By means of setting the annealing temperatures with different temperature gradients, it may be learned that, the different annealing temperatures have a very significant effect on the detection of the BSTD. For example, both the amplification curve and the negative control do not start the curve at the annealing temperatures of 55°C and 55.7°C. At the annealing temperature of 61.4°C, the amplification curve starts the curve at about 16 CT values, a fluorescence value is relatively high, and the amplification curve is good and has no impurity peak. The remaining temperatures can also start a curve, but the fluorescence value is lower than the fluorescence value at the annealing temperature of 61.4°C, and the CT value is also slightly delayed.

Therefore, the annealing temperature of 614°C is selected as the optimal annealing temperature of subsequent experimental conditions.

Embodiment 5 Specificity evaluation of a fluorescent quantitative PCR detection method of BSTD

Bacillus cereus, bacillus subtilis, listeria monocytogenes, escherichia coli and citrobacter freundii are inoculated into an LB broth medium, and are cultured at 37°C to a logarithmic phase for later use. The BSTD B1 is inoculated into a BHI medium, and is cultured at 37°C to the logarithmic phase for later use.

The genomic DNA is extracted by means of the high-temperature pyrolysis method; the concentration and purity of the genome of each strain are measured, so as to determine the purity to be good; and the concentration of each genome is diluted, such that the genome concentrations of all strains are the same. These genomes are used as templates for the SYBR real-time fluorescent quantitative PCR reaction, the M215 is a positive control; and the ddHzO is a negative control. The q-PCR reaction system is shown in Table 1 above, and the

PCR reaction procedure is shown in Table 4 below. Then q-PCR detection is performed separately. LU503460

Table 4 PCR reaction procedure in Embodiment 5 mo

Results of measuring related bacillus cereus, bacillus subtilis and multiple foodborne bacteria are shown in Fig. 5. The primer has a good curve-starting effect only on the positive control M215 and B1 at about 16 Ct values. The bacillus cereus, the bacillus subtilis, the listeria monocytogenes, the escherichia coli and the citrobacter freundii do not start curves, which are consistent with the negative control. Curve starting indicates the accumulation of products during the entire PCR process. If more specific products are present in a sample, amplification is observed in an earlier cycle, and the Ct value is small; if there is less product, amplification is observed in a later cycle, and the Ct value is large; and smaller Ct values indicate a better detection effect.

In conclusion, it may be learned from specificity experiments that, the primer pair and the probe do not start the curve for the bacillus cereus, the bacillus subtilis, the listeria monocytogenes, the escherichia coli and the citrobacter freundii, and only start the curves for the BSTD B1 and the BSTD M215, indicating that the BSTD is highly specific.

Embodiment 6 Sensitivity evaluation of a fluorescent quantitative PCR detection method of BSTD

The BSTD BI is inoculated into a BHI medium, cultured at 37°C to the logarithmic phase for later use and counted. The genomic DNA is extracted by means of the high-temperature pyrolysis method; the concentration and purity of the genomes of the strains are measured, so as to determine the purity to be good; and then the genomes are diluted to 10-9 with 10-fold gradient, which are used as a template for SYBR real-time fluorescent quantitative PCR reaction, and the ddH»O is the negative control. The reaction system and procedure are based on reaction conditions in Embodiment 5, and q-PCR detection is performed.

By means of counting the BSTD B1 cultured to the logarithmic phase, it may be learned that the viable count is 7.5*10°CFU/mL. The sensitivity of the fluorescent quantitative PCR detection method of BSTD is measured. Results are shown in Fig. 6 and show results of the amplification curve of fluorescent quantitative PCR of the genomic DNA template with different concentrations and melting curves. The amplification curves with 7 differeht/503460 concentrations from a stock solution to dilution 10-6 all start the curves within 35 cycles (Ct value<35), and the amplification curves are good. However, the amplification curves with lower concentrations and the negative control all do not start the curves within 40 cycles, indicating that the sensitivity of the method may be detected up to 0.75 CFU/mL. That is to say, if the sample to be detected contains 7-8 BSTD B1 per 10 milliliters, the BSTD B1 may be detected by means of the method.

Embodiment 7 Detection of an artificially-contaminated sample

Bacillus cereus, bacillus subtilis, listeria monocytogenes, escherichia coli and citrobacter freundii are inoculated into an LB broth medium, and are cultured at 37°C a logarithmic phase for later use; and the BSTD B1 is inoculated into a BHI medium, and is cultured at 37°C a logarithmic phase for later use.

In one group, the bacillus cereus, the bacillus subtilis, the listeria monocytogenes, the escherichia coli, the citrobacter freundii and the BSTD B1 are taken 20uL each into 1 mL of sterile ultrapure water; and in the other group, 100 pL of the BSTD BI is taken into 1 mL of sterile ultrapure water.

Then the two groups are vortexed for 1 min by using a vortex mixer, so as to cause the bacterial fluid to be uniformly dispersed; in each group, 1 pL is taken as the template of fluorescent quantitative PCR; and the ddH:O is used as the negative control. The reaction system and procedure are based on the reaction conditions in Embodiment 5, and q-PCR detection is performed.

A detection result of an artificially-contaminated sample is shown in Fig. 7. It may be learned from the figures that, the BSTD B1 contaminated by single bacteria starts a curve at about 18 cycles; the amplification curve contaminated by mixed bacteria starts a curve at 20 cycles; the amplification curve of interference strains that the BSTD B1 is not added is the same as the ddH,O, which both do not start the curve. The concentration of the bacterial fluid contaminated by the single bacteria is higher than the concentration of the bacterial fluid contaminated by the mixed bacteria, such that the amplification curve contaminated by the single bacteria starts the curve several cycles earlier than the amplification curve contaminated by the mixed bacteria. The results show that, the reagent may not only detect the samples contaminated by the BSTD BI, but also detect the specificity of the BSTD B1 in the mixed bacterial fluid of the bacillus cereus, the bacillus subtilis, the listeria monocytogenes, the escherichia coli, the citrobacter freundii and the BSTD B1. That is to say, the reagent may not only detect the samples contaminated by the single bacteria, but also detect the specificity of the BSTD B1 in the samples contaminated by various miscellaneoks/503460 bacteria, such that an application range of the reagent is greatly enlarged.

Embodiment 8 Testing different dairy products on the market 9 different brands of UHT (ultra-high temperature sterilized milk) milk are purchased from a supermarket and labeled as 1 to 9 samples to be detected. In order to simplify the steps of detection operations, 1 uL of milk is directly taken as a template for fluorescent quantitative PCR without pre-treating the milk. The ddH,O is used as the negative control, and the BSTD M215 is used as the positive control. The reaction system and procedure are based on the reaction conditions in Embodiment 5, and q-PCR detection is performed.

The results of determining the UHT milk samples of 9 different brands on the market are shown in Fig. 8. The positive control starts a curve at about 16 cycles, and two product starts a curve at about 27 cycles. Since the milk contains some substances inhibiting the PCR reaction, a typical S-shaped curve is not shown, but the accuracy of the detection results is not affected.

The remaining products and the negative control do not start curves, indicating that the sample 2 and the sample 7 may contain the BSTD.

By means of performing plate spreading on the sample 2 and the sample 7 subsequently, the product contains the BSTD. Then by separating and identifying the strains, and by means of the 16srDNA, it may be learned that, the BSTD in the sample 2 and the sample 7 has 99.6% homology with the BSTD B1 and the BSTD M215. This also shows that the reagent can accurately identify the BSTD in dairy products without complicated operation steps.

It is finally to be noted that, the above embodiments are merely for describing and not intended to limit the technical solutions of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention may be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and shall all fall within the scope defined by the claims of the present invention.

B1-F forward primer sequence: GAGTGCAGAAGAGAAGAG.

LU503460

B1-R reverse primer sequence: GTCAGTTACAGACCAAAGA.

Fluorescence probe sequence: 5’-6-FAM-TTCGCCACTGGTGTTCCTCC-MGB-3".

Claims

Claims: LU503460

1. A PCR amplification primer pair and a probe, wherein an forward primer comprises a sequence shown as SEQIDNO:1, and a reverse primer comprises a sequence shown as SEQIDNO:2; and the probe comprises a sequence shown as SEQIDNO:3.

2. The PCR amplification primer pair and the probe as claimed in claim 1, wherein the probe is a Tagman probe.

3. The PCR amplification primer pair and the probe as claimed in claim 1 or 2, wherein a 3’ terminal of the probe is connected to a fluorescence quenching group, and a 5’ terminal is connected to a fluorescence reporter group.

4. The PCR amplification primer pair and the probe as claimed in claim 3, wherein the fluorescence quenching group is MGB, and the fluorescence reporter group is FAM.

5. A detection reagent, comprising the PCR amplification primer pair and the probe as claimed in any of claims 1 to 4.

6. A detection kit, comprising the PCR amplification primer pair and the probe as claimed in claim 1 or the detection reagent as claimed in claim 5.

7. Applications of the PCR amplification primer pair and the probe as claimed in any of claims 1 to 4 or the detection reagent as claimed in claim 5 or the detection kit as claimed in claim 6 in detection of Bacillussporothermodurans (BSTD).

8. A PCR amplification detection method for Bacillussporothermodurans, comprising: performing PCR amplification on a sample to be detected by using the PCR amplification primer pair and the probe as claimed in any of claims 1 to 4, and during PCR amplification, according to a fluorescence signal, determining whether the sample to be detected contains Bacillussporothermodurans (BSTD); and according to a CT value and a standard curve, quantitatively analyzing the BSTD of the sample to be detected.

9. The PCR amplification detection method for Bacillussporothermodurans as claimed in claim 8, wherein an annealing temperature of a PCR amplification reaction ranges from 55°C to 65°C.

10. The PCR amplification detection method for Bacillussporothermodurans as claimed in claim 8 or 9, wherein the sample to be detected is milk.