KR20120005596A - The simultaneous detection method of pathogenic vibrio spp. using multiplex real-time pcr - Google Patents

Abstract

PURPOSE: A method for simultaneously detecting three kinds of vibrio bacteria causing food poisoning is provided to quickly detect the presence of the vibrio. CONSTITUTION: A method for simultaneously detecting three kinds of vibrio bacteria, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus comprises: a step of performing real time PCR of zot, vmrA, and vuuA genes; and a step of detecting species by melting temperature of PCR products. A complementary primer set encoding the zot, vmrA, and vuuA genes is a sequence set 1,2-3,4-5,6 combination.

Description

The simultaneous detection method of pathogenic Viruo spp. By the method of the method of the detection of pathogenic Vibrio spp. using Multiplex real-time PCR}

The present invention relates to a simultaneous detection method of three Vibrio food poisoning bacteria, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, which are recognized as major food poisoning bacteria in Korea.

The present invention provides a multiplex real-time PCR method targeting zot, vmrA, and vuuA genes for simultaneous detection of V. cholerae, V. parahaemolyticus and V. vulnificus .

Cholerae bacteria ( Vibrio cholerae ), Vibrio parahaemolyticus and Vibrio vulnificus are known to cause gastrointestinal illnesses or septicemia. Ingestion of raw or uncooked food or seafood that has not been properly processed can lead to infection and often fatal consequences. Therefore, the development of rapid, sensitive and reliable detection methods is urgent.

Detections approved by the US Food and Drug Administration (FDA) include most-probable-number (MPN), biochemical assays, and DNA-DNA hybridization methods using selective media. These detections are very specific but take four to five days to complete and are laborious.

Molecular biology, PCR, is an alternative to culture-based methods and has been used as a reliable tool for detecting various pathogens. In particular, multiplex PCR has an advantage of simultaneously detecting multiple targets. However, traditional PCR methods, including multiplex PCR, also require gel electrophoresis for the analysis of amplification products, which are time consuming and labor intensive.

Recently reported real-time PCR is a method to detect the intensity (intensity) of fluorescence generated from amplification products. The result is faster and more sensitive than conventional PCR, and the analysis is very simple. It also eliminates the need for additional experiments such as gel electrophoresis, DNA-DNA hybridization, and sequencing to analyze amplification products.

The present invention has been made in view of the above-mentioned background, and an object of the present invention is to provide the quickest presence or absence of three Vibrio strains of Vibrio cholerae , Vibrio parahaemolyticus , and Vibrio vulnificus . To provide a detection method that can be verified.

In order to detect V. cholerae, V. parahaemolyticus, and V. vulnificus, the present inventors used three PCR primer pairs based on zonular occludens toxin ( zot ) , multidrug efflux pump ( vmrA ) , and vulnibactin outer membrane receptor precursor ( vuuA ) genes. The three PCR primer pairs were designed for real-time PCR to confirm the simultaneous detection of three pathogenic microorganisms.

The present invention as described above is the simultaneous presence or absence of three food poisoning bacteria of cholera bacteria ( Vibrio cholerae ), Vibrio parahaemolyticus and Vibrio vulnificus by using real-time PCR As a detection method, real-time PCR is performed on the zot, vmrA, and vuuA genes of the three bacteria , followed by melting temperature calculated from the melting curve of the PCR product. It is characterized by identifying the target species.

The present invention also provides a nucleotide sequence of a primer set complementary to the DNA sequences encoding the zot, vmrA, and vuuA genes of the cholera bacterium ( Vibrio cholerae ), Vibrio parahaemolyticus and Vibrio vulnificus . The combination is characterized in that the combination of SEQ ID NO: 1,2-3,4-5,6.

In the present invention, for the simultaneous detection of V. cholerae, V. parahaemolyticus, V. vulnificus , a multiplex real-time PCR method targeting zot, vmrA, vuuA genes was developed. Oligonucleotide primers specific for each target strain were designed and quantitatively analyzed by genomic DNA serially diluted 10-fold. The specificity of multiplex PCR was confirmed at different melting temperatures calculated from the melting curves of PCR products obtained from the target species, and sensitivity was determined through linear regression analysis of the C _T value and the amount of target genome per reaction. It was confirmed. The lowest detection limits of the developed detection methods were 9.8 cell / reaction, 9.8 cell / reaction, and 4.1 cell / reaction. This is theoretically close to the maximum sensitivity. The PCR primer set and the optimized real-time PCR method designed in the present invention are expected to provide a valuable tool for the simultaneous detection of pathogenic Vibrio species.

Hereinafter, the present invention will be described in detail with examples.

≪ Materials and methods >

1. Strains and Cultures

In the present invention, the strains used as positive control and negative control are listed in Table 2. V. cholerae and V. parahaemolyticus were passaged in Luria Bertani sodium (LBS) Agar (37 ^o C) and V. vulnificus was passaged in Luria Bertani (LB) Agar (37 ^o C).

Culture of the remaining Vibrio strains followed the method recommended by the culture collection.

Non- Vibrio strains obtained from laboratory stocks were passaged in nutrient agar (37 ^° C).

Extracting genomic DNA to be used as a template for PCR was performed using AccuPrep Genomic DNA Extraction Kit (Bioneer), and ten-fold serial dilution of genomic DNA was performed 10 times to 10 ⁰ to 10 ⁶ .

2. Oligonucleotide primers

PCR primer sequences were designed using the Array Designer for the nucleotide sequences of the genes ( zot, vmrA, vuuA ) in NCBI GenBank (http://www.ncbi.nlm.nih.gov) and NCBI-BLAST (http: / /blast.ncbi.nlm.nkh.gov) confirmed the specificity of the primer on in silico . The search conditions for the primer sequences were used as defaults set by the software manufacturer, and detailed search procedures were also followed by the manufacturer's instructions. Oligonucleotide primer pairs for each target were prepared from the designed primer sequences.

The specificity of the designed primers was verified experimentally by using the aforementioned target strains as positive control and non-target strains as negative control. The PCR reactions used for primer specificity verification included 1 ul (30 ng) of template DNA, 25 μl of Taq PreMix with MgCl ₂ , PCR primers of 1 μl (5 pmole), 1 μl of BSA (20 mg / ml, Bovine Serum Albumin, Roche, Mannheim, Germany) was added to distilled water to 50-μl in total. After initial denaturation at 95 ^o C for 10 minutes, denaturing at 95 ^o C for 15 seconds-Annealing at 55 ^o C for 15 seconds-25 minutes of extension at 72 ^o C and finally 72 ^o Elongation at C was carried out for 20 minutes. PCR thermocycler was used GeneAmp PCR system 9700 (Applied Biosystems, Foster City, USA).

3. Multiplex real-time PCR

Multiplex real-time PCR was optimized with hot-start conditions using SYBR Premix Ex Taq Kit (TaKaRa, Otsu, Shiga, Japan). SYBR Green I was used as the reporter dye and ROX was used as the passive reference dye.

A total of 50 μl of PCR mixture was added to 1 μl template DNA, 25 μl of SYBR Premix Ex Taq, 1 μl ROX, and 1 μl (5 pmole) of PCR primer. ) Was executed within. Thermal-cycling parameter is 55 times with 95 ^o after 10 minutes from the initial shift converter C, 1 bungan elongation at 95 ^o C 15 seconds denaturation -55 ^o C 15 seconds primer binding ^{(annealing) -72 o C (extension} ) The fluorescence signal data was collected in the extension step.

The specificity of Multiplex RTi-PCR was confirmed from different melting temperatures calculated from melting curves of PCR products amplified from each target. After the PCR cycle termination to the Melting curve analysis, at 95 ^o 15 sec at C, 1 min at 60 ^o C, 15 sec at 95 ^o C, 60 ^o C for 15 seconds, the process of detecting the fluorescence signal (fluorescent signal) Was added. Real-time PCR thermocycler was used 7300 Real-Time PCR System (Applied Biosystems, Calif. USA).

Standard curves of real-time PCR were prepared by serial dilution of genomic DNA extracted from V. cholerae (ATCC14035), V. parahaemolyticus (ATCC27519), and V. vulnificus (MO6-24 / O). From the concentration of diluted DNA, the genome size of the target Vibrio species ( V. cholera , 4.08 Mb V. parahaemolyticus , 5.16 Mb V. vulnificus , 5.19 Mb) (http://cmr.tigr.org) and the number of Avogadro (6.02 X 10 ²³ molecules per mole) were used to calculate the genome equivalents (GE), per PCR reaction (6.2X10 ⁰ -6.2X10 ⁶ , 4.1X10 ⁰ -4.1X10 ⁶ , 3.7X10 ⁰ -3.7X10 ⁶ ) Real-time PCR was performed by adding template DNA at GE level. All PCR-amplification reactions were performed by quintuplicate.

Fluorescence signals collected during RTi-PCR were analyzed using Sequence Detection Software ver1.4 (Applied Biosystems, Calif. USA). The signal obtained from the reporter dye was normalized using the signal obtained from the passive reference dye and expressed as a normalized reporter ( Rn ). ΔRn was calculated from the difference between Rn obtained from the PCR reaction including the template and Rn obtained from the initial stage of the PCR reaction (in which the fluorescence signal generated from the PCR amplification product was not different from the background signal). Threshold cycle (C _T ) was calculated as the number of cycles in which the ΔRn value exceeded a statistically significant threshold in the concentration of the PCR product. The threshold value is the average of baseline signals measured between the 3rd and 15th PCR cycles plus 10 standard deviations.

Results and Discussion

1. Specificity of PCR primers and optimization of real-time PCR

PCR primer sets were designed based on the three target gene sequences obtained from the GenBank database (six primers from Table 1, Table 1 are appended here as SEQ ID NOS: 1-6). The specificity of the designed primer pairs was determined by conventional PCR method for the positive control group ( V. cholerae, V. parahaemolyticus, V. vulnificus ) and negative control group (non-target Vibrio species) (Table 2).

PCR products ( V. cholerae 487 bp, V. parahaemolyticus 382 bp, V. vulnificus 155 bp) of the expected size in the positive control were amplified (Fig. 1) from other species belonging to genus Vibrio and from bacteria belonging to other genus. No PCR product was observed.

The thermal cycle parameters of real-time PCR were optimized considering the T _m values of the designed primers. Low melting temperature of designed PCR primers (T _m = zot-f; 54.5 ^o C, zot-r; 53.4 ^o C, vmrA-f; 52.4 ^o C, vmrA-r; 53.7 ^o C, vuuA-f; 56.2 ^o C ^{, vuuA-r; 56.7 o C} ) because ^{standard shuttle cycle (denaturation at 95 o} C and annealing / extension at 60 ~ 66 o C) , if used is was lower the RTi-PCR amplification efficiency 3-step thermal cycle (denaturation at Satisfactory amplification efficiency was obtained at 95 ^o C, annealing at 55 ^o C, and extension at 72 ^o C). Concentrations of MgCl ₂ and SYBR Green I dye were followed by the manufacturer's instructions.

Multiplex real-time PCR showed that the target genomic DNA in all ten-fold dilution concentration ranges (4.4 X 10 ⁶ to 4.4 fg, 5.56 X 10 ⁶ to 5.56 fg, and 5.6 X 10 ⁶ to 5.6 fg, respectively) was positive. ( C _T values) are shown (Fig. 2). The melting temperature of each target amplified product was 87.88 ± 0.164 ^o C, 84.64 ± 0.219 ^o C, and 82.02 ± 0.179 ^o C (Fig. 3). The fluorescence signal of the negative control strains was much lower than the threshold level.

Fig. 3. Sensitivity of Multiplex Real-time PCR

( V. cholerae , 87.88 ^o C; V. parahaemolyticus , 84.64 ^o C; V. vulnificus , 82.02 ^o C).

2. Detection sensitivity

Standard curves to confirm the sensitivity of real-time PCR were prepared by serial dilution of genomic DNA purified from V. cholerae ATCC14035, V. parahaemolyticus ATCC27519, and V. vulnificus MO6-24 / O. Fig. 4). Genome size of each target strain ( V. cholerae , 4.08 Mbp V. parahaemolyticus , 5.46 Mbp V. vulnificus , 5.19 Mbp), (http://cmr.tigr.org) and the number of Avogadro (6.02 X 10 ²³ molecules per mole We calculated that one genome copy (or genome equivalent, GE) of each target genomic DNA was 4.4 fg, 5.56 fg, and 5.6 fg. Multiplex real-time PCR-amplifications are each target genomic DNA was carried out in quintuplicate to ^{6.2 ~ 6.2 X 10 6 GE,} 4.1 ~ 4.1 X 10 6 GE, and 3.7 ~ 3.7 X 10 6 GE.

Minimum detection limits were 6.2 GE, 4.1 GE, and 3.7 GE per reaction, and the corresponding C _T values were 33.16 ± 0.83 ^o C, 33.66 ± 0.91 ^o C, 34.08 ± 0.32 ^o C. The sensitivity of the multiplex real-time PCR technique used in this study is estimated to be increased by about 1000 times (gel electrophoresis after PCR) compared to the detection limit (ca. 10 ³ ) of conventional PCR. In the linear regression analysis of the standard curve plotting the C _T value against the template DNA concentration, the regression coefficient ( r ² ) was 0.99. The slope of the regression plot was -3.29 for V. cholerae, V. vulnificus and -3.39 for V. parahaemolyticus . This amplification efficiency (amplification efficiency) This indicates that the ^{(E = e ln10 / - 1} - slope) respectively 101.3%, 97.2%. In addition, the linear regression analysis of the standard curve plotting the C _T value for the strain diluted 10-fold showed that the regression coefficient ( r ² ) was 0.99 and the slopes were V. cholerae , -3.32; V. parahaemolyticus , -3.43; V. vulnificus , -3.33. Each amplification efficiency was V. cholerae, 100.1%; V. parahaemolyticus, 95.7%; V. vulnificus, 99.7%.

From the above results, it can be expected that the present invention will provide a valuable tool for the simultaneous detection of pathogenic Vibrio species.

<110> Lee Sang Hwan; INDUSTRY-UNIVERSITY COOPERATION FOUNDATION HANKUK UNIVERSTIY OF FOREIGN STUDIES <120> The simultaneous detection method of pathogenic Vibrio spp. using          Multiplex real-time PCR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gagaggcggc ggagatag 18 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 attgtctacg aggcgataac g 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggtgttgtgt tcgtggtatt g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cttggatgct cggttctact g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcgagcacca aacatgacag 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gagcattagc gacccaataa gc 22

Claims (2)

After real-time PCR of the zot, vmrA, and vuuA genes of cholera bacteria ( Vibrio cholerae ), Vibrio parahaemolyticus and Vibrio vulnificus , Characterized by identifying the target species by the melting temperature calculated from the melting curve of the PCR product (product),
Simultaneous detection of three food poisoning bacteria, Vibrio cholerae , Vibrio parahaemolyticus and Vibrio vulnificus , using real-time PCR.
The method of claim 1,
Cholera bacteria ( Vibrio cholerae ), Vibrio enterobacteriaceae The base sequence combination of the primer set complementary to the DNA sequences encoding the zot , vmrA , and vuuA genes of parahaemolyticus ) and Vibrio vulnificus is a combination of the sequence listing sets 1,2-3,4-5,6 Characterized by
Cholera bacteria (Vibrio cholerae), Vibrio Vibrio bacteria (Vibrio DNA using real-time polymerase chain reaction (real-time PCR) parahaemolyticus ) and Vibrio vulnificus (three types of food poisoning bacteria) simultaneous detection of the presence or absence.