High-throughput molecular detection method for water-mediated protozoa and / or worm

Abstract

The invention discloses a high-throughput molecular detection method of water-mediated protozoa and / or worms, which comprises the following steps: designing primer probes of 22 detection targets based on an OpenArray chip, preparing positive control plasmids, extracting DNA of an environment sample to be detected, preparing the OpenArray chip for sample adding, carrying out real-time fluorescent PCR detection, and determining the content of the water-mediated protozoa and / or worms according to the Ct value of the measured species. And determining the types and concentrations of water-mediated protozoa and worms contained in the environment sample to be detected. By adopting the method disclosed by the invention, high-throughput molecular sieve detection can be simultaneously carried out on 22 kinds of water-medium protozoa and worms, and the detection efficiency is remarkably improved; according to the method disclosed by the invention, the types and the concentrations of the water-medium protozoa and the worms in the environment are detected, and the detection result is high in accuracy and credibility; moreover, the method has high repeatability, stability and detection sensitivity on a detected target, and the credibility of a detection result is good.

Description

Technical Field

[0001] The present invention relates to a high-throughput molecular detection method for microorganisms, in particular to a high-throughput molecular detection method for hydrozoan protozoa and / or worms based on OpenArray chips, and belongs to the field of environmental monitoring. Background Art

[0002] The greatest risk to public health in water is microorganisms, and water is a common medium for the spread of many pathogens. Waterborne pathogens can be divided into three categories: bacteria, viruses, and parasites, the latter of which includes protozoa and worms. Protozoa refers to a group of single-celled eukaryotic organisms, including sporozoans (intracellular parasites), flagellates (have tail-like structures for movement), amoebas (use temporary cell body protrusions as pseudopodia), and ciliates (move by beating multiple hair-like structures called cilia).

[0003] Many waterborne protozoa and helminths are zoonotic, meaning they can infect both humans and animals. They are usually present in low concentrations in the environment, but can still pose a considerable public health risk by causing waterborne infectious diseases, particularly in the elderly, children and people with acquired immunodeficiency syndrome.

[0004] Cryptosporidium is a significant cause of childhood mortality in developing countries. Giardia lamblia (G. lamblia) (also known as Giardia duodenalis) contaminates water supplies worldwide, and ingestion of its cysts can lead to giardiasis, an acute, self-limited gastroenteritis. Although most protozoan outbreaks are caused by Cryptosporidium and Giardia (the “two worms”), which are listed in the National Drinking Water Hygiene Standards, other protozoan species such as Toxoplasma gondii, Entamoeba histolytica, Microsporidia, Acanthamoeba, and Blastocystis hominis, as well as some helminths, have also been associated with waterborne outbreaks. Contamination of drinking or recreational water with these protozoa and helminths increases the risk of waterborne diseases, but little information is available, and this is closely related to the current high-throughput detection methods for protozoa and helminths.

[0005] At present, the detection methods for pathogenic parasites such as protozoa and helminths at home and abroad mainly include microscopic detection of single species, real-time quantitative PCR (qPCR), nested PCR, flow cytometry, loop-mediated isothermal amplification (LAMP), etc. There are currently no related articles and patents on high-throughput detection methods for water-borne protozoa and helminths.

[0006] A major disadvantage of microscopic testing is that it cannot identify the type of parasite. Due to the limitations of the optical system, the resolution of microscopic testing is limited. Microscopic testing requires a well-trained operator to correctly operate the microscope and identify the microorganisms in the sample. Microscopic testing usually takes a lot of time to prepare samples, observe and identify protozoa, which is time-consuming and labor-intensive for large-scale analysis or for complex samples.

[0007] Real-time quantitative PCR (qPCR) has a low throughput. Each experiment quantifies one or two targets. When the sample size and the number of detection targets are large, it takes a lot of time and increases the complexity of the experiment.

[0008] Flow cytometry is very complex and expensive to operate. It is usually used to detect the number and size of cells. For some small protozoa, additional steps or techniques may be required to enhance the sensitivity and accuracy of its detection. For unknown or uncommon protozoa, customized markers and dyes may be required, further increasing the complexity and cost of the experiment.

[0009] LAMP requires the design of multiple primers, including four specific primers and two loop primers. The design of these primers is relatively complex and needs to take into account the characteristics of the target sequence and the interaction between primers, which increases the difficulty of the experiment. The LAMP amplification product is usually a mixture, including DNA fragments of different lengths and structures, and the experimental results are not easy to analyze. Summary of the invention

[0010] The present invention mainly aims at the technical problems that the detection results of pathogenic parasites such as protozoa and worms based on microscopic detection, quantitative qPCR, nested PCR, flow cytometry, and loop medium isothermal amplification are poor in accuracy and sensitivity; time-consuming and labor-intensive; the operation method is complicated, the experimental difficulty is high, the analysis of the experimental results is difficult, the detection steps are cumbersome, the detection cost is high; the stability of the detection method is poor, and the like. A high-throughput molecular detection method for water-borne protozoa and / or worms based on an OpenArray chip is provided. The method of the present invention designs primer probes for detecting target species of 22 water-borne protozoa and worms based on the OpenArray chip, performs real-time fluorescence PCR detection, and can realize the simultaneous detection of 22 target species of 192 samples, greatly shortens the detection time, significantly improves the detection efficiency, reduces manual operation, reduces manual operation errors, has a fast detection speed, and has a high accuracy of the detection result, and realizes the high-throughput molecular detection of water-borne protozoa and worms.

[0011] To achieve the purpose of the present invention, the present invention provides a high-throughput molecular detection method for water-borne protozoa and / or helminths, comprising the following steps:

[0012] 1) Design primers and probes

[0013] Select target genes based on the specific genes of the target species to be detected, and design primers and probes through Primer;

[0014] 2) Preparation of positive control plasmid

[0015] First, based on the target gene of the target species selected when designing primers and probes in step 1), the first 20 bp of the forward primer is cut as the starting position of the target gene prepared by the positive plasmid; the last 20 bp of the reverse primer is cut as the ending position of the target gene prepared by the positive plasmid, and the target gene prepared by the positive plasmid corresponding to each target species is obtained; then, the target gene, 16S rRNA gene, and 18S rRNA gene prepared by the positive plasmid corresponding to each target species are cloned into an Escherichia coli cloning vector to obtain a positive plasmid;

[0016] 3) Extracting DNA from the collected environmental samples to obtain DNA from environmental samples to be tested for future use;

[0017] 4) Preparation and loading of OpenArray chips

[0018] 4-1) First, according to the format of the gene expression chip, load the names of the samples to be tested and the location information of each sample on the 96-well plate in the downloaded Sample Tracker software; then, use the Sample Tracker software to correspond the names and location information of the samples on the 96-well plate to the 384-well plate (384-well plate exclusive to OpenArray chip); then, use the Sample Tracker software to export the file of the sample arrangement mode (i.e. sample layout mode) of each sample to be tested in the 96-well plate and 384-well plate, and record it accurately;

[0019] 4-2) Add the samples to be tested into the corresponding wells of the 96-well plate according to the sample arrangement (sample layout method) derived in step 4-1);

[0020] 4-3) Adding Real-Time PCR Master Mix (i.e., OpenArray reaction premix) to the 384-well plate dedicated to the OpenArray chip; then, according to the sample loading arrangement derived in step 4-1), adding the sample DNA to be tested in the 96-well plate to the corresponding small wells in the 384-well plate; sealing the film to prepare the sample loading 384-well plate;

[0021] 4-4) placing the sample-loaded 384-well plate prepared in step 4-3) on the 384 module of the AccuFill instrument, placing the OpenArray chip on the Plate Holder of the AccuFill instrument, wherein the barcode side of the Open Array chip is placed on the left side of the Plate Holder facing upward; turning on the AccuFill instrument, transferring the sample to be tested on the sample-loaded 384-well plate to the OpenArray chip, and preparing the sample-loaded OpenArray chip;

[0022] 5) Real-time fluorescence PCR detection

[0023] The sample-loaded OpenArray chip prepared in step 4) is placed in an Applied Biosystems QuantStudio 12KFlex System instrument for real-time fluorescence quantitative PCR detection to determine the Ct value of the target gene of each species contained in the sample, thereby obtaining the types and contents of protozoa and helminths in the environmental sample.

[0024] Among them, the target species to be detected in step 1) are 22 kinds of cytosporidial protozoa and worms, specifically: Giardia lamblia, Giardia lamblia, Cryptosporidium, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium melitensis, Entamoeba histolytica, Naegleria fowleri, Acanthamoeba, Acanthamoeba cabotseni, Acanthamoeba castellanii, Acanthamoeba polyphaga, Cyclospora, Toxoplasma gondii, Blastocystis hominis, Enterosporidium pyogenes, Encephalitis microsporidia, Isospora belli, Balantidium coli, Schistosoma, Guinea worm, Echinococcus.

[0025] In particular, the primers and probes for detecting 22 species of protozoa and helminths are:

[0026]

[0027]

[0028] In particular, the 5′-terminal fluorescent group (i.e., fluorescent dye) of the probe in step 1) is as follows:

[0029]

[0030] In particular, the fluorescent group at the 5′-end of the probe is preferably 6-FAM with a maximum emission peak of ˜520 nm, and the quenching group at the 3′-end is MBG.

[0031] In particular, in step 2), the positive plasmids corresponding to each target species are prepared by enzyme digestion to obtain the target gene, 16S rRNA gene, and 18S rRNA gene, which are cloned together into the Escherichia coli cloning vector PUC57 to obtain the positive control plasmid.

[0032] In particular, the 16S rRNA gene is a 16S rRNA gene of bacteria; and the 18S rRNA gene is a 18S rRNA gene of eukaryotes.

[0033] In particular, the positive plasmid was synthesized by Thermofisher.

[0034] The positive control plasmid of the present invention includes 24 specific gene fragments of the detection target, that is, the target gene, 16S rRNA gene and 18S rRNA gene prepared by the positive plasmid corresponding to 22 target species. The concentration of the prepared positive plasmid is 1×10 7 copies / μL.

[0035] In particular, in step 3), a DNA extraction kit is used to extract DNA from the collected environmental samples.

[0036] In particular, the DNA extraction kit is an existing and known DNA extraction kit in the art, such as DNeasy tissue kit (adding liquid nitrogen freezing and boiling water thawing and ultrasonic process), QIAamp DNA Stool Minithe kit, Mag Extractor-Genome kit, Quick-DNA Faecal / Soil Microbe kit, Epicentre MasterPure Complete (MPC) DNA and RNA Purification kit or SPINfor Soil DNA Kit.

[0037] In particular, if the environmental sample collected in step 3) is a water sample, the environmental water sample is first concentrated and enriched by using calcium carbonate flocculation method, and the DNA of the water sample is extracted after the concentrated sample is obtained.

[0038] In particular, the environmental water sample is concentrated and enriched by the calcium carbonate flocculation method according to the following steps:

[0039] A) adding CaCl2 solution (1 mol / L, 100 ml) and NaHCO3 solution (1 mol / L, 100 ml) to the collected water sample in sequence under stirring, and adjusting the pH of the water sample by adding NaOH solution (10 mol / L) and / or HCl solution (3 mol / L) to the water sample; flocculating the protozoa and worms in the sample under the condition that the pH of the water sample is maintained at 10±0.05;

[0040] B) After flocculation treatment for at least 30 minutes, the mixture was allowed to stand for 12-16 hours; the supernatant was then removed by siphoning to obtain the protozoa and worm flocculation precipitate (referred to as "protozoa-worm" flocculation precipitate);

[0041] C) adding sulfamic acid solution (mass percentage concentration 10%, 200 mL) to the "original-vermicompost" flocculation precipitate, stirring, and performing redissolution treatment, so that the "original-vermicompost" flocculation precipitate is dissolved to obtain the "original-vermicompost" reconstituted solution;

[0042] D) Use a centrifuge to centrifuge the "original-vermicular" reconstituted solution (2000 g, 10 min); discard the supernatant to obtain the centrifugal precipitate, namely the "original-vermicular" centrifugal precipitate (concentrated sample).

[0043] In particular, the samples to be tested in step 4-1) are environmental test samples, positive control plasmids, and negative control RNase-free water.

[0044] In particular, the sample layout is the name of the sample to be tested and the position of the sample to be tested in the 96-well plate and the 384-well plate.

[0045] In particular, in step 4-2), the amount of DNA of the corresponding sample to be tested added to each well of the 96-well plate is 5 μl.

[0046] In particular, the concentration of the environmental test sample DNA is 20±1 ng / μl; the amount of the corresponding environmental test sample DNA added to each well is 5 μl, the positive control is 5 μl, and the negative control is 5 μl.

[0047] In particular, the volume ratio of the sample DNA to be tested added to each well of the 384-well plate in step 4-3) to the OpenArray chip reaction premix is ​​1:1.

[0048] In particular, the amount of sample DNA to be tested added to each well of the 384-well plate is 2.5 μl; the volume of the OpenArray reaction premix added is 2.5 μl.

[0049] In particular, the Real-Time PCR Master Mix (ie, OpenArray reaction premix) contains a mixture of DNA polymerase, dNTP and other substances.

[0050] In particular, each OpenArray chip in step 4-4) has 48 sub-arrays, and each sub-array includes 64 through holes.

[0051] In particular, the primers and probes corresponding to the target species to be detected are fixed in the small holes of each subarray of the OpenArray chip described in step 4-4).

[0052] Specifically, a primer and probe for a target species to be detected are fixed in a small well of the OpenArray chip.

[0053] In particular, in each subarray of the OpenArray chip, the primers and probes of each target species to be detected are fixed in at least two small holes.

[0054] In particular, 4 small holes are left unused at the sample inlet and sample outlet of each subarray of the OpenArray chip, and the primers and probes corresponding to the target species to be detected designed in step 1) are fixed in the other 56 small holes.

[0055] In particular, the 16S rRNA gene and the 18S rRNA gene are fixed in the small holes of each subarray of the OpenArray chip.

[0056] In particular, in each subarray of the OpenArray chip, the 16S rRNA gene and the 18S rRNA gene are each fixed in two small holes.

[0057] In particular, the real-time fluorescence quantitative PCR detection reaction conditions in step 5) are: pre-denaturation: 50°C, 2 minutes; 95°C, 10 minutes; denaturation: 95°C, 15 seconds; annealing and extension: 60°C, 60 seconds; 40 cycles.

[0058] Compared with the prior art, the molecular detection method of the present invention has the following advantages and benefits:

[0059] 1. The detection efficiency of the pathogenic parasite detection method based on OpenArray chip of the present invention is high. The method of the present invention can simultaneously perform high-throughput molecular screening on 19 species of hydrozoan protozoa and 3 species of helminths, and the detection efficiency is significantly improved;

[0060] 2. The method of the present invention is used to detect the types and concentrations of water-borne protozoa and helminths in the environment. The detection results are highly accurate and reliable. The correlation coefficient R of the standard curve for detecting 22 types of water-borne protozoa and helminths established according to the method of the present invention is2 The range of is 0.983-0.998, the increase ratio of the copy number of the target DNA molecule in each cycle of real-time fluorescence PCR amplification is high, and the range of the amplification efficiency E reaches 80-109%, that is, the amplification efficiency of the high-throughput detection of the method of the present invention is between 80-109%, indicating that the detection result of the method of the present invention is accurate and reliable and can truly reflect the actual situation.

[0061] 3. The method of the present invention is used to detect the types and concentrations of hydrozoans and helminths in the environment, and has good repeatability and stability for the detection target, and the detection result is highly reliable.

[0062] 4. The detection method of waterborne protozoa and helminths of the present invention has a fast detection speed and high accuracy of the detection result, thus realizing high-throughput detection of waterborne protozoa and helminths.

[0063] 5. The method of the present invention can simultaneously detect 22 target species in 192 samples, greatly shortening the detection time and significantly improving the detection efficiency.

[0064] 6. The method of the present invention has less manual operation, thus reducing the error of manual operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a schematic diagram of an arrangement of primers and probes of target species to be detected in a subarray in an OpenArray chip, where each small grid represents each small hole in the subarray. DETAILED DESCRIPTION

[0066] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0067] The present invention uses equipment:

[0068] AccuFill instrument; Applied Biosystems QuantStudio 12K Flex System instrument; Sample Tracker software are all from Thermo Fisher Scientific, USA.

[0069] Example 1 Primer and probe design

[0070] The species information of 22 species of hydrozoans and helminths detected by the high-throughput method of the present invention is shown in Table 1.

[0071] (1) Design primers and probes:

[0072] The target gene was selected through NCBI, and the target gene was selected according to the specific gene of the target species to be detected (i.e., the 22 protozoa and worms of the present invention), that is, the gene that is different between different target species. Primers and probes were designed by Primer premierv6.24, and the primer TM value was optimized to ensure that the primer TM value was between 58-62°C, the difference in the TM value of the upstream and downstream primers did not exceed 2°C, the primer length was controlled at 9-40bp, and the GC content was between 30-80%; the first base at the 5' end of the probe could not be G, and the probe design avoided excessive repetition of the base G, especially 4 or more in a row; the designed primers and probes for molecular detection of the target species to be detected (22 water-borne protozoa and worms) are shown in Table 1.

[0073] In the present invention, the 5′-end fluorescent group of the probe is 6-FAM with a maximum emission peak of 520 nm, and the quenching group at the 3′-end is MBG.

[0074] Fluorescent dyes are used to emit fluorescent signals. The strength of the fluorescent signals indicates the amount of species. The present invention uses 6-FAM fluorescent dye with a maximum emission peak of ∼520 nm.

[0075]

[0076]

[0077] The 5′-end fluorescent group of the present invention, except 6-FAM, other fluorescent groups are as follows, all of which are applicable to the present invention; when other fluorescent groups are used, the quenching group at the 3′-end can absorb the fluorescence of the corresponding fluorescent group.

[0078] The following fluorescent dyes are suitable for the 5′-terminal fluorescent group (fluorescent dye) of the probe of the present invention:

[0079]

[0080] The 6-FAM fluorescent dye with a maximum emission peak of ∼520 nm is preferably selected as the reporter fluorescence.

[0081] Example 1A Preparation of positive plasmid

[0082] 1. Screening positive plasmids to prepare target genes

[0083] Based on the target genes of each species selected when designing primers and probes in Example 1, the first 20 bp of the forward primer was cut as the starting position for preparing the positive plasmid target gene; the last 20 bp of the reverse primer was used as the ending position for preparing the positive plasmid target gene; the positive plasmids corresponding to 22 species were screened to prepare the target gene;

[0084] 2. Gene cloning

[0085] The target gene, 16S rRNA gene and 18S rRNA gene prepared by the 22 screened positive plasmids were integrated into the Escherichia coli cloning vector (PUC57) to obtain positive plasmids, wherein the 16S rRNA gene is the 16S rRNA gene of bacteria; and the 18S rRNA gene is the 18S rRNA gene of eukaryotes.

[0086] The positive plasmid was prepared by enzyme digestion and synthesized by Thermo Fisher Scientific, USA. There was only one positive plasmid in the whole experiment. The prepared positive plasmid included 24 specific gene fragments of the detection target, including the target genes, 16S rRNA gene and 18S rRNA gene of 22 species screened in step 1). The concentration of the prepared positive plasmid was 1×10 7 copies / μL.

[0087] Example 2 Preparation of OpenArray chip

[0088] 1. File download

[0089] 1A. Download the Plate Setup File (.spf / .tpf) file, which contains the embedding site ID, location information and arrangement on each OpenArray chip. Download the .spf / .tpf file for each chip according to the serial number of each chip. Download URL: https: / / www.thermofisher.com / cn / en / home / products-and-services / product-types / downloadopenarray-tpf-and-spf-plate-files.html;

[0090] 1B. Download the Sample Information File (.csv): This file is generated by the Sample Tracker software and contains the names and arrangement information of the samples to be tested loaded onto the chip.

[0091] 1C. Template file (.edt) file: chip running experiment template, including experiment type, site information, sample information, etc. (generated before the experiment);

[0092] 1D, experiment file (.eds): Experimental running results.

[0093] 2. Sample addition

[0094] 2A. In Sample Tracker software (Thermo Fisher Scientific, USA), according to the format of the gene expression chip, load the names of the samples to be tested (environmental samples to be tested, positive controls (i.e., positive control plasmids) and negative controls (i.e., RNase-free water)) and their location information on the 96-well plate in the downloaded Sample Tracker software; then use Sample Tracker software to map the sample information (name, location information) on the 96-well plate to the 384-well plate (384-well plate exclusive to OpenArray chips); then export the file of the sample DNA loading arrangement in the 96-well plate and 384-well plate, i.e., the sample layout file, and record it accurately;

[0095] 2B. In a 96-well plate, according to the sample arrangement (sample layout file) exported in the software, add sample DNA (about 20 ng / μl), positive control, and negative control to the corresponding wells of the 96-well plate. The amount of sample DNA added to each well is 5 μl, and the amount of positive control plasmid and negative control is 5 μl;

[0096] 2C. Use a dispenser to add 2.5 μl of Real-Time PCR MasterMix (i.e., OpenArray reaction premix, a mixture of substances such as DNA polymerase and dNTP) to the 384-well plate dedicated to the OpenArray chip; then, according to the sample arrangement of the sample layout table exported from the software, use a dispenser to draw 2.5 μl of the sample DNA in the 96-well plate and add them to the corresponding small holes in the 384-well plate; the volume ratio of the sample DNA added to the 384-well plate to the reaction premix is ​​1:1, and the amount of the added volume is determined by the chip layout.

[0097] OpenArray reaction premix, OpenArray chip, OpenArray chip-specific consumables, and OpenArray chip-specific 384-well plate were all purchased from Thermo Fisher Scientific, USA.

[0098] Record the serial number of the OpenArray chip for use when downloading the SPF file.

[0099] OpenArray chip exclusive consumables (Accessories Kit, 384-well plate, aluminum film for sealing 384-well plate, gun tip), shotgun (1.5-2.5μl)

[0100] 2D. Seal the 384-well plate with an aluminum film, shake and mix, and centrifuge (1000 rpm, 1 min) to obtain a 384-well plate with samples.

[0101] 3. OpenArray chip loading

[0102] The samples on the 384-well plate were transferred to the OpenArray chip. After instrument detection, the Ct detection values ​​of the target species contained in the sample, the Ct values ​​of each target species contained in the positive control plasmid, and the negative control Ct values ​​were obtained. Each chip has a total of 3072 (48×64) microwells, and the volume of each microwell is 0.039μL. The instrument detection time is 2.5 hours.

[0103] 3A. Take out the OpenArray chip and thaw and equilibrate it at room temperature for 30 minutes. Take out the oil-containing syringe, remove the cover, install the accompanying gun tip, and place it on a clean table for use.

[0104] 3B. Open and run QuantStudio TM AccuFill software;

[0105] 3C. Place the 384-well plate prepared in step 2 on the AccuFill instrument (tear off the aluminum film after installing the 384-well plate to avoid liquid splashing). The AccuFill software divides the 384-well plate into 8 areas, and marks the area where the current sample exists on the AccuFill software; place the OpenArray chip on the Plate Holder of the AccuFill instrument, with the barcode side of the Open Array chip facing up on the left side of the Plate Holder;

[0106] After the OpenArray chip is opened, the chip can be loaded on Accufill within 1 hour.

[0107] In a specific embodiment of the present invention, the arrangement of primers and probes in the wells of each subarray on the OpenArray chip is as follows: Figure 1 shown.

[0108] Each OpenArray chip has 48 sub-arrays, each sub-array includes 64 through-holes, and a total of 3072 through-holes.

[0109] The injection port and the sample outlet of the OpenArray chip are the locations where the injection starts and the reaction ends. Because the real-time fluorescence PCR reaction is prone to instability, 4 small holes are left unused at the injection port (4 holes on the lower right) and the sample outlet (4 holes on the lower left). Figure 1 ), and primers and probes for detecting different species are fixed in the small holes at other positions.

[0110] The primers and probes designed in Example 1 for detecting 22 species (ie, 22 species of protozoa and helminths) are fixed in the small holes of each subarray on the OpenArray chip, and the primers and probes for detecting each species are fixed in at least two small holes.

[0111] The 16S rRNA gene quality control and 18S rRNA gene quality control are also fixed in the small holes of each subarray on the OpenArray chip, and the 16S quality control and 18S quality control are fixed in at least 2 small holes respectively.

[0112] In the specific implementation mode of the present invention, the arrangement of primers and probes is in accordance with Figure 1 Except for the arrangement in the manner of , other arrangements are applicable to the present invention.

[0113] 3D, close the Accufill instrument door, click load, and load the chip; when the OpenArray chip is loaded, open the instrument door, remove the OpenArray chip, and then seal the chip according to the OpenArray chip sealing operation manual to obtain the sample chip (i.e., the OpenArray chip loaded with sample DNA). The sealing steps are as follows:

[0114] Remove the protective film (red side) on the cover of the OpenArray chip; place the OpenArray chip on Place the cover on the Plate Press, with the end of the OpenArray chip with the QR code on the left; then place the cover on the OpenArray chip, and press the lever for about 20 seconds;

[0115] Use the prepared syringe to inject oil into the chip from the small hole on the back of the chip. It is best to complete it within 60 seconds. The injection process should be continuous and not too fast, otherwise the sample will be flushed out of the through hole. Try to reduce the generation of bubbles. Small bubbles will not affect the experiment.

[0116] Hold the chip vertically, Plug the chip and turn it clockwise until the handle of the plug falls off automatically.

[0117] Spray the dust-free paper with alcohol to wipe the OpenArray chip clean, and then wipe the alcohol clean with a clean filter paper.

[0118] 4. Real-time fluorescence PCR detection

[0119] Remove the protective film on the surface of the sample-loaded OpenArray chip (i.e., the OpenArray chip loaded with sample DNA) and place the sealed OpenArray chip into QuantStudio TM On-board testing on the 12K Flex System instrument.

[0120] Real-time fluorescence quantitative PCR was performed using the Applied Biosystems QuantStudio 12K Flex System instrument to determine the Ct value of the target gene of each target species contained in the sample.

[0121] Reaction conditions: pre-denaturation (50°C, 2 min; 95°C, 10 min), denaturation (95°C, 15 sec), annealing and extension (60°C, 60 sec) for 40 cycles.

[0122] After loading, the chip should be sealed within 90 seconds to prevent excessive liquid evaporation.

[0123] Example 3 Drawing a standard curve

[0124] 1. Prepare positive plasmid dilution

[0125] The positive plasmid stock solution (1×10 7 copies / μL, purchased from Thermo Fisher Scientific, USA) for gradient dilution, and the final concentrations were 1×10 6 ,1×10 5 ,1×10 4 ,1×10 3 ,5×10 2 The positive plasmid was diluted with 5 concentration gradients of 10 copies / μL.

[0126] The positive plasmid was prepared according to the method of Example 1A. The positive plasmid was synthesized by ThermoFisher Scientific (USA) according to the enzyme digestion method.

[0127] 2. Preparation of standard curve using OpenArray chip

[0128] 2-1) Download files

[0129] The same as step 1 "file download" in Example 2.

[0130] 2-2) Sample addition

[0131] The method is the same as step 2 "sample loading" of Example 2, except that the samples to be tested loaded on the 96-well plate in the downloaded Sample Tracker software are respectively five dilutions of positive plasmids with concentration gradients; and during the actual operation of the experiment, the five dilutions of positive plasmids with concentration gradients are added to the actual 96-well plate according to the sample loading arrangement exported in the software.

[0132] 2-3) Chip loading

[0133] The same as step 3 "chip loading" in Example 2.

[0134] 2-4) Real-time fluorescence PCR detection

[0135] The same as step 4 "real-time fluorescence PCR detection" in Example 2.

[0136] That is, using the Applied Biosystems QuantStudio 12K Flex System instrument, real-time fluorescence quantitative PCR detection was performed on the sample chip to determine the Ct value of the target gene of each species in 5 concentration gradients of positive plasmid dilutions.

[0137] The measurement results are shown in Table 2.

[0138] Table 2 Concentration and Ct value of positive plasmid dilution

[0139]

[0140] The results showed that within a certain range, the Ct value of the real-time fluorescence PCR detection of each species in the positive plasmid dilution was linearly related to the logarithm of the concentration of the positive plasmid dilution (base 10). The logarithm of the concentration of the positive plasmid dilution (unit: copies / μL) was used as the abscissa, and the Ct value of the real-time fluorescence PCR detection of the target gene of each species in the positive plasmid dilution with different concentrations was used as the ordinate to draw a curve to obtain the standard curve corresponding to each species. The standard curve is shown in formula (1):

[0141] y = kx + b (1)

[0142] In formula (1), y is the Ct value of each species in the positive plasmid dilution; x is the logarithm of the concentration of the positive plasmid dilution (10 is the base); k is the slope; and b is the intercept.

[0143] The standard curves of 22 species and 2 quality controls drawn according to the assay results are shown in Table 3.

[0144] A total of 24 standard curves were established for this method, including 19 protozoa, 3 worms, and two quality controls, 18S and 16S. The correlation coefficients of the standard curves were R 2 The range of is 0.983-0.998, the range of amplification efficiency E is 80-109%, and the established equation is shown in Table 4. The results show that the correlation between the fluorescence curve of the established detection system and the target concentration is good and the accuracy is high.

[0145] Amplification efficiency: refers to the ratio of the increase in the number of copies of the target DNA molecule in each cycle in the PCR reaction. Theoretically, the number of copies of the target DNA molecule should double in each cycle, but in actual reactions, due to the influence of various factors, the amplification efficiency usually does not reach 100%. The amplification efficiency is calculated by the slope of the standard curve. Amplification efficiency control between 80%-120% is considered to be an acceptable error range. Amplification efficiency within this range can ensure the reliability of experimental results.

[0146] The amplification efficiency of the high-throughput detection of the method of the present invention is between 80% and 109%, indicating that the experimental results of the method of the present invention are accurate and reliable and can truly reflect the actual situation.

[0147] Table 3 Standard curve, correlation coefficient and amplification efficiency of detection targets

[0148]

[0149]

[0150] Example 4 Preparation of DNA samples to be tested

[0151] 1. Sample collection

[0152] The samples are divided into water samples, soil samples, and organic fertilizer samples, among which:

[0153] Water sample collection: Use a 10L plastic bucket to collect water samples; Soil and organic fertilizer sample collection: According to the five-point sampling method, collect samples from five locations and mix them as one sample to improve the representativeness of the sample.

[0154] After collection, soil and organic fertilizer samples were frozen at -20°C before DNA extraction.

[0155] The collected water samples need to be concentrated and enriched;

[0156] 2. Sample enrichment

[0157] The present invention adopts a calcium carbonate flocculation method to carry out concentration and enrichment processing of samples, wherein the calcium carbonate flocculation method includes two parts: flocculation and re-dissolution.

[0158] 2A. Flocculation treatment

[0159] The solutions used in the flocculation process include: 1 mol / L CaCl2 solution, 1 mol / L NaHCO3 solution, 10 mol / L NaOH solution, and 3 mol / L HCl solution, wherein the amount of CaCl2 solution added is 100 mL, and the amount of NaHCO3 solution added is 100 mL. The pH is adjusted to 10±0.05 using NaOH solution and HCl solution.

[0160] Under stirring, add CaCl2 solution (1 mol / L, 100 ml) and NaHCO3 solution (1 mol / L, 100 ml) to the water sample (10 L) collected in step 1) in sequence, stir while adding, and measure the pH of the sample at the same time, and adjust the pH of the sample to be tested by adding NaOH solution (10 mol / L) and HCl solution (3 mol / L) so that the pH of the sample is maintained at 10±0.05; perform flocculation and precipitation treatment on protozoa and worms in the sample;

[0161] During the flocculation process, a stirrer is used for stirring, and the stirring time is greater than 30 minutes. After the stirring is completed, the sample to be tested is allowed to stand for 12-16 hours, and the supernatant is removed by siphoning to obtain the protozoa and worm flocculation precipitate, referred to as the "protozoa-worm" flocculation precipitate, which is set aside.

[0162] The amount of NaOH and HCl added is not fixed. You need to test the pH value of the sample while adding it. If the pH is higher than 10±0.05, you need to use HCl to lower the pH value.

[0163] 2B. Redissolution

[0164] Add sulfamic acid solution (10% (mass percentage concentration), 200 mL) to the "original-vermicompost" flocculation precipitate, stir, and perform redissolution treatment, so that the "original-vermicompost" flocculation precipitate is dissolved to obtain the "original-vermicompost" reconstituted solution;

[0165] The reconstituted solution was centrifuged in a centrifuge at a speed of 2000 g for 10 min. The supernatant was discarded to obtain a centrifugal precipitate, namely, the "original-vermicular" centrifugal precipitate.

[0166] The centrifugal precipitate of "original-vermicular" was stored in a refrigerator at -20℃ for later use.

[0167] 3. Extract sample DNA

[0168] A DNA extraction kit was used to extract the DNA of the centrifugal precipitate of "original-worm" to obtain the sample DNA to be tested; then NanoDrop One / OneC (Thermo Fisher Scientific, Waltham, USA) was used to detect the purity and concentration of the sample DNA; and then the DNA sample to be tested was stored in a -20°C refrigerator for future use.

[0169] In the present invention, the DNA concentration during the general detection should be controlled at 20ng / ul. If the sample DNA concentration is higher than 20ng / ul, it needs to be diluted to about 20ng / ul before use.

[0170] The kits used for DNA extraction in the present invention are as follows: DNeasy tissue kit (adding liquid nitrogen freezing and boiling water thawing and ultrasonic process), QIAamp DNA Stool Minithe kit, Mag Extractor-Genome kit, Quick-DNA Faecal / Soil Microbe kit, Epicentre MasterPure Complete (MPC) DNA and RNA Purification kit, SPIN for Soil DNA kit was used to extract DNA according to the operating steps and requirements in the kit instructions.

[0171] Example 5 Sample detection (real-time fluorescence PCR)

[0172] 1. Collect environmental testing samples

[0173] Three types of samples collected from source water, urban sewage treatment plants and livestock and poultry feces were tested for protozoa and helminths, including:

[0174] Drinking water source samples (n=46); sludge samples from municipal sewage treatment plants (n=12); fecal samples from pigs, chickens, sheep and cattle (n=10).

[0175] 2. Environmental sample enrichment

[0176] The collected sludge samples and feces samples were frozen at -20℃.

[0177] The collected drinking water source samples (46 samples) were concentrated and enriched in the same manner as step "2. Sample enrichment" in Example 4 to obtain "raw-vermicular" centrifugal precipitates of the drinking water source samples.

[0178] 3. Extract sample DNA

[0179] The DNA of the 68 collected environmental samples was extracted in the same manner as step “3. Extracting sample DNA” in Example 4, and the DNA samples of the 68 environmental samples obtained were stored in a -20°C refrigerator for future use.

[0180] 4. Preparation of OpenArray Chip

[0181] 4-1) Download files

[0182] The same as step 1 "file download" in Example 2.

[0183] 4-2) Sample addition

[0184] The same as step 2 "sample loading" of Example 2 except that the samples to be tested loaded on the 96-well plate in the downloaded Sample Tracker software are the 68 sample DNAs obtained in step 3), the positive control plasmid and the negative control; in the actual operation of the experiment, the 68 sample DNAs are added to the actual 96-well plate according to the sample arrangement exported in the software.

[0185] That is, the sample DNA (about 20 ng / μl) extracted in the same manner as step “3. Extracting sample DNA” in Example 4, the positive control plasmid prepared in Example 1A, and the negative control were added to the corresponding wells of a 96-well plate, respectively. The amount of the corresponding sample DNA added to each well was 5 μl, and the amount of the positive control plasmid and the negative control were both 5 μl;

[0186] 4-3) Chip loading

[0187] The same as step 3 "chip loading" in Example 2.

[0188] 5. Real-time fluorescence PCR detection

[0189] The same as step 4 "real-time fluorescence PCR detection" in Example 2.

[0190] Place the sealed OpenArray chip loaded with sample DNA in the QuantStudio TM Real-time fluorescence PCR amplification and Ct value detection were performed on a 12K Flex System instrument, where the reaction conditions included 40 cycles of pre-denaturation (50°C, 2 minutes; 95°C, 10 minutes), denaturation (95°C, 15 seconds), annealing and extension (60°C, 60 seconds).

[0191] The Ct value results of real-time fluorescence PCR detection in environmental samples are shown in Tables 4, 5, and 6.

[0192] According to the Ct value of the target species detected, the types and quantities of the target species contained in the environmental samples were counted, and the detection rate of the target species in the samples was calculated. The detection rate results of protozoa and helminths in environmental samples are shown in Table 7.

[0193] From the test results of Tables 4, 5, 6 and 7, we can see that:

[0194] Using a high-throughput qPCR detection method, 17 of the 22 target species (hydroprotozoa, helminths) were detected in three environmental samples (a total of 68 samples, including drinking water sources (n=46), municipal sewage treatment plant sludge (n=12), and livestock and poultry feces (n=10)). Among them, the three species with the highest detection rates were Acanthamoeba (50.0%, i.e., Acanthamoeba was detected in 34 of the 68 samples), Acanthamoeba castellanii (11.8%, i.e., Acanthamoeba was detected in 8 of the 68 samples), and Enterosporidium bieneusi (11.8%).

[0195] The distribution of protozoa and helminths differed between the three media types: 13 species were detected in drinking water sources, 13 species were detected in municipal wastewater treatment plant sludge, and 11 species were detected in livestock and poultry manure.

[0196] In drinking water sources, the most frequently detected target species were Acanthamoeba, Naegleria fowleri and Acanthamoeba castellani; in municipal sewage treatment plant sludge, the dominant target species were Acanthamoeba, Enterosporidiosis and Acanthamoeba polyphaga; the most frequently detected species in livestock and poultry feces were Balantidium coli, Acanthamoeba castellani and Enterosporidiosis. Cryptosporidium, Cyclospora, Enterosporidiosis, Balantidium coli and Isospora belli were detected in drinking water sources, municipal sewage treatment plant sludge and livestock and poultry feces.

[0197]

[0198]

[0199]

[0200] In Table 7, “-” means not detected

[0201] Example 6 Repeatability Test

[0202] 1. Preparation of DNA positive plasmid for intra-group and inter-group difference detection

[0203] The positive plasmid stock solution (1×10 7copies / μL, purchased from Thermo Fisher Scientific, USA) for gradient dilution to obtain 1×10 5 , 1×10 4 The positive plasmids with 100 copies / μL DNA were used to verify the intra-group and inter-group differences of the high-throughput detection method of hydrozoan and helminth of the present invention.

[0204] Intra-group differences refer to the differences in results obtained when the same OpenArray chip is used to detect the same target; inter-group differences refer to the differences in results obtained when different OpenArray chips are used to detect the same target.

[0205] 2. Preparation of OpenArray chips for intra-group and inter-group difference detection

[0206] 2-1) Download files

[0207] The same as step 1 "file download" in Example 2.

[0208] 2-2) Sample addition

[0209] In addition to loading samples on two 96-well plates in the downloaded Sample Tracker software, each 96-well plate is loaded with 1×10 5 , 1×10 4 copies / μL of DNA positive plasmid; in the actual operation of the experiment, 1×10 5 , 1×10 4 The steps were the same as step 2 “sample loading” of Example 2 except that 100 copies / μL of DNA positive plasmid were added into two actual 96-well plates.

[0210] 2-3) Chip loading

[0211] The same as step 3 "chip loading" in Example 2.

[0212] 2-4) Real-time fluorescence PCR detection

[0213] The same as step 4 "real-time fluorescence PCR detection" in Example 2.

[0214] That is, using the Applied Biosystems QuantStudio 12K Flex System instrument, real-time fluorescence quantitative PCR detection was performed on the sample chip to measure 1×10 5 , 1×10 4 The Ct value of the target gene of each species in the DNA positive plasmid with 10 copies / μL.

[0215] Reaction conditions: pre-denaturation (50°C, 2 min; 95°C, 10 min), denaturation (95°C, 15 sec), annealing and extension (60°C, 60 sec) for 40 cycles.

[0216] 1×10 5 , 1×10 4 The average Ct value and standard deviation (SD) corresponding to the positive plasmid copies / μL DNA are shown in Table 8.

[0217] Table 8 Average Ct value and standard deviation results of positive plasmids in repeatability test

[0218]

[0219]

[0220] The coefficient of variation (CV) of intra-group and inter-group difference detection of each target species was calculated according to formula (1). The calculation results are shown in Table 9, where formula (1) is as follows:

[0221] Coefficient of variation (CV) = (standard deviation SD / mean value) × 100% (1)

[0222] The standard deviation and average value in formula (1) are the standard deviation and average value of the Ct values ​​measured by real-time fluorescence PCR.

[0223] Since there are 22 target species and 2 quality control (16S rRNA gene, 18S rRNA gene) positive controls, the data in Table 9 of this example are the ranges of Ct values ​​and CV values ​​of the 24 species.

[0224] Table 9 Repeatability test results

[0225]

[0226] The concentration of positive plasmid was 10 5 copies / μL, group C t The coefficient of variation (CV) ranged from 1.0 to 4.1% between groups. t The coefficient of variation CV of the values ​​ranged from 1.2 to 5.5%;

[0227] The concentration of positive plasmid was 10 4 When C t The coefficient of variation (CV) ranged from 1.7% to 4.6% between groups. t The coefficient of variation CV of the values ​​ranged from 3.0 to 6.4%, all less than 10%, indicating that the method had good repeatability and stability for the detection target.

[0228] The above embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. A high-throughput molecular detection method for hydrozoan protozoa and / or helminths, characterized in that: The steps include: 1) Select the target gene based on the specific gene of the target species to be detected, and design primers and probes through Primer; 2) Based on the target gene of the target species selected when designing primers and probes in step 1), the first 20 bp of the forward primer of the corresponding target species is cut as the starting position of the target gene prepared by the positive plasmid; the last 20 bp of the reverse primer is cut as the ending position of the target gene prepared by the positive plasmid, and the target gene prepared by the positive plasmid corresponding to each target species is obtained; then: the target gene, 16S rRNA gene, and 18S rRNA gene prepared by the positive plasmid corresponding to each target species are cloned into the Escherichia coli cloning vector to obtain a positive control plasmid; 3) Extracting DNA from the collected environmental samples to obtain DNA from environmental samples to be tested for future use; 4) Preparation and loading of OpenArray chips 4-1) First, according to the format of the gene expression chip, load the names of the samples to be tested and the location information of each sample on the 96-well plate in the downloaded Sample Tracker software; then, use the Sample Tracker software to correspond the names and location information of the samples on the 96-well plate to the 384-well plate (384-well plate exclusive to OpenArray chip); then, use the Sample Tracker software to export the file of the sample arrangement mode (i.e. sample layout mode) of each sample to be tested in the 96-well plate and 384-well plate, and record it accurately; 4-2) Add the samples to be tested into the corresponding wells of the 96-well plate according to the sample arrangement (sample layout method) derived in step 4-1); 4-3) Adding Real-Time PCR Master Mix (i.e., OpenArray reaction premix) to the 384-well plate dedicated to the OpenArray chip; then, according to the sample loading arrangement derived in step 4-1), adding the sample DNA to be tested in the 96-well plate to the corresponding small wells in the 384-well plate; sealing the film to prepare the sample loading 384-well plate; 4-4) placing the sample-loaded 384-well plate prepared in step 4-3) on the 384 module of the AccuFill instrument, and placing the OpenArray chip on the Plate Holder of the AccuFill instrument, wherein the barcode side of the Open Array chip is placed on the left side of the Plate Holder facing upward; turning on the AccuFill instrument, transferring the sample to be tested on the sample-loaded 384-well plate to the OpenArray chip, and preparing the sample-loaded OpenArray chip; 5) placing the sample-loaded OpenArray chip prepared in step 4) in an Applied Biosystems QuantStudio 12KFlex System instrument, performing real-time fluorescence quantitative PCR detection, and determining the Ct value of the target gene of each species contained in the sample, thereby obtaining the types and contents of cytomegaloviruses and helminths in the environmental sample.

2. The high-throughput molecular detection method according to claim 1, characterized in that: The target species to be detected in step 1) are 22 kinds of cytosporidial protozoa and helminths, specifically: Giardia, Giardia lamblia, Cryptosporidium, Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium meleagridis, Entamoeba histolytica, Naegleria fowleri, Acanthamoeba, Acanthamoeba cabotsonii, Acanthamoeba castellanii, Acanthamoeba polyphaga, Cyclospora, Toxoplasma gondii, Blastocystis hominis, Enterosporidium pyogenes, Encephalitis microsporidia, Isospora belli, Balantidium coli, Schistosoma, Guinea worm, Echinococcus.

3. The high-throughput molecular detection method according to claim 1 or 2, characterized in that: The primers and probes for detecting 22 species of protozoa and helminths are:

4. The high-throughput molecular detection method according to claim 1 or 2, characterized in that: If the environmental sample collected in step 3) is a water sample, the environmental water sample is first concentrated and enriched by using a calcium carbonate flocculation method, and the DNA of the water sample is extracted after the concentrated sample is obtained.

5. The high-throughput molecular detection method according to claim 1 or 2, characterized in that: The samples to be tested in step 4-1) are environmental test samples, positive control plasmids, and negative control RNase-free water.

6. The high-throughput molecular detection method according to claim 1 or 2, characterized in that the steps The volume ratio of the sample DNA to be tested added to each well of the 384-well plate in 4-3) to the OpenArray chip reaction premix is ​​1:

1.

7. The high-throughput molecular detection method according to claim 1 or 2, characterized in that: The primers and probes corresponding to the target species to be detected are fixed in the small holes of each subarray of the OpenArray chip described in step 4-4).

8. The high-throughput molecular detection method according to claim 1 or 2, characterized in that: In each subarray of the OpenArray chip, the primers and probes of each target species to be detected are fixed in at least two small holes.

9. The high-throughput molecular detection method according to claim 1 or 2, characterized in that: The real-time fluorescence quantitative PCR detection reaction conditions in step 5) are: pre-denaturation: 50°C, 2 minutes; 95°C, 10 minutes; denaturation: 95°C, 15 seconds; annealing and extension: 60°C, 60 seconds; 40 cycles.

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