rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay - food grade absorbent pad
The rapid high-
For the control of food safety, the throughput detection of food pathogens is crucial.
In this study,channel up-
Conversion fluorescence technology
Based on side flow (TC-UPT-LF)
A rapid and simultaneous detection of 10 popular food-derived pathogens was established.
Ten different kinds of singletarget UPT-
LF bars are developed and integrated into a TCUPT-
LF disc with optimization.
No concentrated TC-UPT-
The detection sensitivity of LF assay for each pathogen was rmbcfu ML-1 or 5 cfu ML-1, and the sample was 10 cfu/0 After enrichment. 6u2009mg.
The determination also shows good linearity, allowing for quantitative detection, the linear fitting coefficient of the determination (R2)of 0. 916–0. 998.
10 detection channels are not crossed
Therefore, multiple targets can be specifically detected.
When 279 real food samples were tested, the results were highly consistent (100%)with culture-based methods.
The results of 110 food samples artificially contaminated with a single or multiple target showed a high detection rate (≥80%)
For most target bacteria.
In general, TC-UPT-
LF method allows rapid, quantitative and simultaneous detection of common food pathogens in 10 foods within 20 min, especially for rapid detection and monitoring of food pathogens in food and water foods.
Monoclonal antibody (mAbs)
The target bacteria used in this study were prepared. Eight-week-
Obtained old female male/c mice from the experimental animal research center of the Academy of Military Medical Sciences (China).
The mouse acquisition was approved by the Ministry of Health of the General Logistics Department of the Chinese People's Liberation Army.
All the experiments were carried out in accordance with the welfare and ethical guidelines of experimental animals in China.
All experimental programs have been approved by the experimental animal welfare and ethics committee of the Beijing Institute of Microbiology and Epidemiology (Beijing, China). UCP (NaYF:Yb/Er)
From Shanghai keruen fluorescence Technology Co. , Ltd. , the diameter of about 50 nm is obtained. Ltd (Shanghai, China).
The peaks of excitation and emission spectra are 541 nm and nm, respectively.
5 nm, respectively.
Cellulose film (SHF 1350225)
It was purchased from the company. (
Bedford, MA, USA). Papers (470 and 903)
Used to make sample pads and absorption pads are obtained from Schleicher and Schuell(Keene, NH, USA).
TC-laminated card and plastic boxUPT-
The lf cd is designed by our group and manufactured by Shanghai Liangxin Biotechnology Co. , Ltd. (Shanghai, China)
Shenzhen jincanhua Industrial Co. , Ltd. (SHENZHEN, China), respectively.
Including Taiwan pan peptide, yeast extract powder, salt and three (hydroxymethyl)
Aminometane, HCl, NaHPO, KHPO and SDS are all analytical grade, from Sigma-Aldrich (St. Louis, MO, USA).
Skim milk powder was purchased from a local supermarket.
01 and O139 were obtained from the China Center for Disease Control and Prevention (Beijing, China);
8 Other target pathogens are kept in our laboratory.
Alkaline protein powder water (
20g L, NaCO. 12HO 0.
2 u2009 g · L, nacl5 u2009 g · L, KNO 0. 1u2009g L [pH 8. 4])
For the cultivation of 01 and O139, LBS broth (
20g L of Taiwan Pan, 10g L of yeast extract powder and 10g L of salt [pH 7. 2])
Used for eight other bacteria.
All bacteria grow overnight at 37 °c and shake at 200 rpm. The TC-UPT-
LF sensors are designed and manufactured by our laboratory and Shanghai Institute of optical precision machinery, Chinese Academy of Sciences (Shanghai, China).
Different from single
The channel UPT sensor, which contains a highprecision two-
The size disk platform that allows straight and rotating motion of the disc as well as movement of the channel.
Therefore, it can capture the signal of 10 channels of the CD continuously. The UPT-
The LF bars used in the study are based on
Antibody sandwich immune assay
Initially, the binding pad was fixed with a ucp-monoclonal antibody binding (
1 mg mL, 30 μ l cm).
Use the corresponding monoclonal antibody (
2 mg mL, 1 μ l cm)
For the target bacteria on the test track (T lane)
And the goat.
Mouse IgG antibody (
2 mg mL, 1 μ l cm)
In the control Lane (C lane).
Then assemble the sample pad, the bonding pad, the cellulose film and the suction paper as shown. The TC-UPT-
The LF disc contains 10 channels and can contain 10 different singletarget UPT-
LF article, developed as described above.
The strips in the disk overlap on the side of the sample pad.
Liquid samples can be evenly distributed to 10 channels simultaneously and evenly through drainage parts (glass fiber)
Located in the center of the disc.
Treatment buffer with bacterial solution and optimized general sample before detection (0. 05u2009M Tris-HCl [pH 8. 0]containing 2.
5% skim milk powder and 0. 25% SDS)
Mix thoroughly in a ratio of 1: 9. An aliquot (1. 4u2009mL)
Then add the mixture to the disc.
After 15 minutes, scan the signal from the disc via TC-UPT-LF biosensor.
Complete signal reading and reporting within 2 minutes.
The Peak area of the test Lane and control Lane is called "t value" and "c value" respectively ".
The T/C ratio is then calculated and treated as a result.
T/C is higher than the cut sampleoff value (
Mean value of blank T/C ratio ± 3SD)
People who are considered to be positive and the T/C ratio is lower than the cut value
From sample processing to results, the whole operation is completed within 20 minutes.
Harvest bacterial cultures and store them in sterile saline (0.
85% salt water)
The final concentration is 1.
0 × 10 cfu mL was determined by plate counting method.
Dilute the standard solution of each target pathogen with phosphate buffer (0. 02u2009M NaHPO, 0. 01u2009M KHPO [pH 7. 2])
Report on the final accounts of 0 u2009 × 10u2009 CFU mL. 0u2009×u200910u2009CFU mL.
All standard bacterial solutions and blank controls (
Tested with TCUPT-
Three LF analyses.
As a comparison, these solutions are also related to the corresponding singletarget UPT-LF strips.
The minimum concentration of the target pathogen that can be detected positively is considered to be the detection sensitivity of this assay.
Each target of quantitative bacteria construction and logging curve (T/C -cut-off)on the -axis and log(
Concentration of bacterial solution 【CFU mL])on the -
Shaft, with OriginPro 8. 0.
Linear fitting coefficient determined (R)
Used to evaluate quantitative linearity.
Specificity of each detection channel of TC-UPT-
In the case of a concentration of 1, LF plates were evaluated separately with nine other food source bacteria.
0 × 10 cfu mL as described above.
10 different target bacteria were prepared in one solution (
The final concentration is 1.
0 × 10 cfu mL for each target)
Assess the capacity of the TC-UPT-
LF analysis of multiple targets is detected at the same time.
Influence of non
Evaluation of sensitivity and specificity of multiple analyses with continuous concentration of target bacterial solution target bacteria (
0cfu cfu mL to 1. 0u2009×u200910u2009CFU mL)
, Mixed with 9 other pathogens (
The final concentration of each drug is 1. 0u2009×u200910u2009CFU mL).
We received 279 food samples from Shanghai.
Entry-Exit Inspection and Quarantine Bureau (Shanghai, China)
It includes dairy products, seafood, drinks, snacks and meat.
Pre-test, weigh and homogenize food samples with manual sterility and culture in 5 ml LBS broth (
Sample with 700 μ l liquid, 0.
6 mg solid sample)
About 14 Thanh and then test as described above.
Standard methods for culture, separation and identification of bacteria are used. Food samples (
From negative samples in real food samples)
10 pathogens, including 100, were also prepared for artificial contamination
The target sample contains one of 10 target bacteria and all 10 target bacteria in 10 samples.
Because food samples may be contaminated with trace amounts of pathogens (