CN113512606A

CN113512606A - Honeycomb chip-based kit for high-throughput detection of intestinal protozoa and detection method

Info

Publication number: CN113512606A
Application number: CN202010275854.6A
Authority: CN
Inventors: 姜岩岩; 曹建平; 沈玉娟; 袁忠英
Original assignee: National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention
Current assignee: National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention
Priority date: 2020-04-09
Filing date: 2020-04-09
Publication date: 2021-10-19

Abstract

The invention discloses a honeycomb chip-based kit and a detection method for detecting intestinal protozoa in high flux, wherein a microfluidic system is integrated on a honeycomb chip and used for detecting cryptosporidium, giardia, microsporidian and amoeba histolytica. The invention solves the complex steps of carrying out multiple PCR detections, can simultaneously detect multiple genes by one reaction, defines the action of pathogens, greatly lightens the workload, shortens the detection time and reduces the detection cost.

Description

Honeycomb chip-based kit for high-throughput detection of intestinal protozoa and detection method

Technical Field

The invention relates to the technical field of agriculture and animal quarantine, in particular to a detection method of cryptosporidium, giardia, microsporidia and amoeba histolytica, and particularly relates to a multiplex PCR detection method and a kit for detecting various intestinal protozoa in high flux based on a honeycomb chip. Furthermore, the invention relates to specific primers for the simultaneous detection of cryptosporidium, giardia, microsporidia and histolytica amoeba.

Background

Cryptosporidium, Giardia lamblia (Giardia lamblia for short), Microsporosis cubensis and Amiba histolytica are new intestinal protozoa which are commonly suffered by human beings and animals, are mainly transmitted by water, cause consumption diarrhea, are important public health problems facing the world and are one of the most difficult problems facing the human beings so far; can be fulminating in epidemic and cause serious public health accidents. The infection of people is mainly caused by taking drinking water and food polluted by oocysts/cysts and the like, and the caused diseases take diarrhea as main clinical symptoms, are distributed globally and are one of the main causes of death of AIDS patients. Infection of people with normal immune function can cause acute and self-limiting diarrhea, infection of infants, people with immune function deficiency or immunosuppression, chronic consumption diarrhea, and even death. The diseases can be transmitted from animals to humans, from humans to animals, and widely transmitted from animal to animal and human to human, causing great economic impact. Worldwide, children under 250 ten thousand years of age 5 die each year from diarrhea caused by infection with intestinal pathogens, including intestinal protozoa.

The cryptosporidium infection rate of China is 0.5-13.5%, the giardia infection rate is 2.52%, the amoeba infection rate is 3.55-4.5%, and the microsporidian infection rate is 0.23-13.49%. Cryptosporidiosis, which was listed by the WHO as one of six global diarrheas, was increasingly attracting attention in 1986 as a suspected indicator of AIDS (AIDS) patients and by the WHO and the U.S. CDC as a new infectious disease, and was listed as the third highest on the us government bioterrorism list (https:// wwn. CDC. gov/foodborneoutbreaks/default. aspx), the only parasitic pathogen among them. Meanwhile, cryptosporidium is also one of ten pathogens for monitoring food-borne diseases of FoodNet in the United states, and ranks second; the cryptosporidium and giardia are one of the essential water quality indexes of the sanitary Standard for Drinking Water in China and are also the etiology of national infectious disease monitoring technology platform monitoring, which is a major scientific and technological project in China. There are data showing that cryptosporidiosis can be caused to spread epidemic with only 1 oocyst.

Giardiasis is listed as one of the 10 major parasitic diseases that endanger human health worldwide. The World Health Organization (WHO) classified it as a disease that is easily overlooked (ND) in 2004. The first disease example of giardiasis in China is reported by Kessel and the like in 1924, and the giardia infection rate of a human body is reduced from 2.52% of national parasite survey of 1 st time (1988-1992) to 0.60% of national parasite survey of 3 rd time (2014-2015). In rural areas, the pollution source is mainly feces of sick people or animals, sewage of the human or animals and water supply system pipelines which are repaired for a long time, and the secondary pollution of drinking tap water is caused by that sewage is sucked into the pipelines by the leakage of the pipe walls. In cities, the filtration disinfection process of drinking water is mainly unable to completely eliminate giardia cysts in water, i.e. tap water purified by using filtration equipment can still pass through the filter membrane and exist in drinking water because the cysts can change shapes. Results from studies with bright, etc. adult populations of 5 raw and 9 tap waters indicated that: detecting giardia in raw water, wherein the density is L-45/10L; the detection rate of Giardia in tap water is 22%, and the density is 1/100L.

About 5000 million people worldwide infect endolytic amoeba, 4-11 million people die of amoeba disease every year, and the death rate is second to malaria and schistosomiasis among parasitic diseases and is third place. At present, the infection rate of the amoeba in lysostaphin of the population in China generally shows a descending trend, and the infection rate is reduced from 0.949 percent when the national parasite survey is carried out for the 1 st time (1988-1992) to 0.06 percent (including the amoeba in Dispa) of the 3 rd time (2014-2015). But the positive rate of the anti-entamoeba histolytica antibody in some provincial and jurisdictional populations is 1.06% to 14.39%; the positive rate of the anti-tissue-soluble entamoeba antibody in the serum of HIV/AIDS patients in some provinces of China is obviously higher than that of non-HIV infected patients.

Microsporidiosis is the most common microsporidiosis of the Pediobolus pipiensis in humans, which is distributed worldwide. In 1959, Subayasi et al reported the first human infection cases in Japan, demonstrating the pathogenicity of microsporidian in humans. The infection rate of HIV positive diarrhea patients in developed countries is 2% -78%; in developing countries, the infection rates of adults with HIV positive diarrhea and children are 2.5% -51% and 17.4% -76.9% respectively. In 1995, the human microsporidian infection cases in China were found for the first time in hong Kong, and in 1997, the human cases were found again in Guangzhou. In 2011, cases of human enterobacter peelii infection are found for the first time in Jilin province, so far, human infection has been reported in 8 provinces (directly prefectured cities) including Heilongjiang, Henan, Jilin, Henan, Hubei, Chongqing, Guangxi, Yunnan and Shanghai, investigation populations mainly comprise diarrhea populations and AIDS patients, and the infection rate is 0.2% -22.5%.

At present, the detection aiming at cryptosporidium, giardia and amebiasis is mainly morphologically, and is easy to miss detection and false detection. The epidemiological detection of microsporidian is mainly based on common PCR detection. However, the lack of rapid detection tools, no detection kit and no high-throughput detection technology seriously restrict the monitoring and research on the new intestinal protozoa.

The microfluidics subject developed in the 90 s of the 20 th century refers to the scientific technology for operating fluid in tiny network channels (5-500 microns), is the development and fusion of various modern technologies such as molecular biology technology, micromachining technology, mechanical manufacturing technology, computer technology and the like, is a micro device based on the principle of massively processing biological information molecules in parallel, and has the characteristics of large information flux, automation and systematization; the micro-fluid chip is used for operation and transmitting micro-liter (10 mu L) to nano-micro-liter (10m mu L) fluid, can integrate a plurality of steps of biochemical reaction including sample loading, reaction, detection, analysis and the like on one or a plurality of micro-fluid chips, has micro-channel pore size only in micron order, has the functions of concentration and enrichment, can accelerate the reaction and shorten the test time, thereby greatly reducing the test cost. Compared with the conventional experimental technology, the technology greatly reduces the consumption of reagents (at least 3 orders of magnitude), and simultaneously generates little waste liquid during analysis; the energy transfer and the material dispersion in a micro range are faster and more uniform, the heat energy conduction is fast, and various operations and controls are easier to realize, so the reaction is fast, the yield is high, the pollution is less, and the cost is low. At present, microfluidic chips have been developed from separation detection to high-function full-analysis systems including pretreatment of complex samples; from analytical tools, to microchemical reaction and synthesis means including on-line detection. The molecular nucleic acid analysis is carried out on the microfluidic chip, the analysis capability of the microfluidic chip is combined with the specificity of nucleic acid reaction, the defect of cross contamination of nucleic acid can be effectively overcome, the reaction efficiency is improved, the operation steps are simplified, the detection time is shortened, and the consumption of samples, reagents and energy is greatly reduced. The micro-fluidic chip for molecular nucleic acid analysis uses micro-processing technology to establish micron-scale immunoreaction space, and accelerates reaction kinetics process due to size reduction, so that immunoreaction speed based on biomacromolecule diffusion control is improved by several orders of magnitude. The combination and integration of multiple functions of the microfluidic chip also make molecular biological detection on the microfluidic chip have more potential advantages compared with conventional nested PCR nucleic acid detection, and therefore, the microfluidic chip is receiving more and more attention.

With the development of molecular biology technology, the detection and identification of the intestinal protozoa can be realized based on different molecular markers. However, the detection of a single insect species is expensive, time-consuming and labor-consuming. A novel isothermal Nucleic acid amplification (LAMP) technique suitable for gene diagnosis was disclosed in journal of Nucleic Acids Res by Notomi, a Japanese scholarly in 2000. The technology is successfully applied to detection of diseases such as SARS, avian influenza, HIV and the like, and in the event of H1N1 influenza A in 2009, Japan Rongyan chemical company (hereinafter referred to as "Rongyan company") receives an invitation from WHO to complete development of a H1N1 loop-mediated isothermal amplification method detection kit, and plays a positive role in preventing rapid spread of the disease through early rapid diagnosis. The technology has been widely applied to disease detection, food and cosmetic safety inspection and import and export rapid diagnosis caused by various viruses, bacteria, parasites and the like in Japan, and has been accepted by European and American countries. The technology has the advantages of high specificity and high sensitivity, simple operation, low requirement on instrument and equipment, simple result detection, no need of gel electrophoresis like PCR (polymerase chain reaction), judgment of the result of the loop-mediated isothermal amplification reaction by observing the generation of white turbidity or green fluorescence by naked eyes, simplicity, convenience and quickness, and suitability for basic level rapid diagnosis. Is a novel nucleic acid amplification method, and is characterized in that 4 specific primers are designed aiming at 6 regions of a target gene, under the action of strand displacement DNA polymerase (Bst DNA polymerase), the nucleic acid amplification of 10^9 to 10^10 times can be realized within 15 to 60 minutes after the constant temperature amplification is carried out at the temperature of 60 to 65 ℃, and the method has the characteristics of simple operation, strong specificity, easy detection of products and the like. Upon DNA synthesis, pyrophosphate ions precipitated from deoxyribonucleic acid triphosphate substrates (dNTPs) reacted with magnesium ions in the reaction solution to generate a large amount of magnesium pyrophosphate precipitate, which appeared white. Therefore, the turbidity can be used as an index of the reaction, and whether amplification is carried out or not can be identified only by observing the white turbid precipitate with naked eyes without complicated electrophoresis and ultraviolet observation. Because the loop-mediated isothermal amplification reaction does not need a PCR instrument and expensive reagents, the method has wide application prospect. Because the technology has high sensitivity, the problem of easy aerosol pollution caused by uncovering exists, most laboratories in China cannot be strictly partitioned at present, and the problem of false positive is serious.

Therefore, the invention is improved in the aspect of reducing pollution, and a microfluidic system is integrated on a honeycomb chip, so that the high-throughput rapid detection is facilitated on one hand, and primers are anchored on the chip and provided with microfluidic capillaries on the other hand, so that the pollution is reduced. In the detection of intestinal protozoa, the method still stays in the single tube detection pathogen of LAMP, and the preparation of related gene chips aiming at the intestinal protozoa by using a solid phase carrier is not carried out. The invention can simultaneously detect a plurality of intestinal protozoa in a stool sample and is an efficient detection method aiming at the identification of cryptosporidium subspecies.

Disclosure of Invention

One of the technical problems to be solved by the invention is to provide a honeycomb chip-based kit for detecting various intestinal protozoa in a high-throughput manner. The invention establishes a rapid, sensitive and specific detection kit by taking four protozoa, namely cryptosporidium, giardia, amoeba and microsporidian, as research objects.

The second technical problem to be solved by the invention is to provide a detection method of microfluidic coupled LAMP for four protozoa, namely, cryptosporidium, giardia, amebic tissue and nosema peelii. The synthesis of specific primers of four intestinal protozoa LAMP solves the complicated steps of carrying out multiple PCR detection, one-time reaction can achieve the effect of simultaneously detecting multiple genes and defining pathogens, thereby greatly reducing the workload, shortening the detection time, reducing the detection cost and having greater development trend.

The invention aims to solve the technical problem of providing a method for detecting intestinal protozoan molecular biological nucleic acid, which is a nucleic acid high-throughput detection method based on a capillary microarray and is used for improving the detection throughput and the detection efficiency, and reducing the detection cost and the sample consumption.

The invention is realized by the following technical scheme:

in the first aspect of the invention, a honeycomb chip-based kit for high-throughput detection of intestinal protozoa is provided, which is integrated on a honeycomb chip by adopting a microfluidic system and is used for detecting cryptosporidium, giardia, microsporidian and entamoeba histolytica, and the kit comprises any one or any combination of the following 8 groups of primers:

group 1, primers specific for Cryptosporidium (CRY):

CRY-F3: 5′-CTTACTCCTTCAGCACCTTA-3′，SEQ ID NO.1；

CRY -B3: 5′-CAAGAAAGAGCTATCAATCTGT-3′，SEQ ID NO.2；

CRY -FIP: 5′-CGTCAATTCCTTTAAGTTTCAGCCTGAGAAATCAAAGTCTTTGGGTT-3′，SEQ ID NO.3；

CRY -BIP:5′-CCTGCGGCTTAATTTGACTCACAATCCTTCCTATGTCTGGAC-3′，SEQ ID NO.4；

CRY -LF: 5’-TGCGACCATACTCCCCCCA-3’，SEQ ID NO.5；

CRY -LB: 5’-ACACGGGAAAACTCACCAG-3’，SEQ ID NO.6；

group 2, primers specific for human cryptosporidium (Ch):

Ch-F3: 5’-GGCAATCAGGTTGAGTCA-3’ ，SEQ ID NO.7;

Ch-B3: 5’-CGGTATAGAAAGCACTATCGT-3’ ，SEQ ID NO.8;

Ch-FIP:5’-AGGCAAACAAATCGACGGTTG-AGATCAAGAAGATCACTCACA-3’ ，SEQ ID NO.9;

Ch-BIP: 5’-ACCCTTAATGGTGGTAAGAGAATTGCAACCAAACTGTACTTGTCTC-3’ ，SEQ ID NO.10;

group 3, primers specific for Cryptosporidium parvum (Cp):

Cp-F3: 5’-TCGCAC CAGCAA ATA AGG C-3’ ，SEQ ID NO.11;

Cp-B3: 5’-GCCGCA TTC TTC TTT TGGAG-3’ ，SEQ ID NO.12;

Cp-FIP: 5’-ACCCTGGCTACCAGAAGCTTCAGAACTGGAGACGCAGAA-3’ ，SEQ ID NO.13;

Cp-BIP:5’-GGCCAAACTAGTGCTGCTTCCCGTTTCGGTAGTTGCGCCTT-3’ ，SEQ ID NO.14；

Cp-LF: 5’-GTACCACTAGAATCTTGACTGCC-3’ ，SEQ ID NO.15；

Cp-LB: 5’-AACCCACTACTCCAGCTCAAAGT-3’ ，SEQ ID NO.16；

group 4, primers specific for cryptosporidium turkey (Cm):

Cm-F3：5’-CCTTTGAAAACGAATCAAGTTCT-3’ ，SEQ ID NO.17;

Cm- B3：5’- CAATTCTTTTACCACCTTGGA-3’ ，SEQ ID NO.18;

Cm-FIP：5’-GTCATTTTCTGTTGGGGAATTTGAATACAATAAAAATCAAGGTTGACG-3’ ，SEQ ID NO.19 ;

Cm-BIP: 5’-GTCTGAGGAAAGCTTGTCTAGATCAGCACATCAACAGTTCCAG-3’ ，SEQ ID NO.20；

group 5, primers specific for cryptosporidium andersi (Ca):

Ca-F3: 5’-CGTGCAAAGAGAACACTTTC-3’ ，SEQ ID NO.21；

Ca-B3: 5’-CCTACTAATACAACATCATGTACT-3’ ，SEQ ID NO.22；

Ca-FIP: 5’-TCCTCAAATCTTGCACGACTTATWGATTCATCTACWCAAGCAACAAT-3’ ，SEQ ID NO.23；

Ca-BIP: 5’-GTTCTGATTATTTCCGTGGCACACTCTTATCCATTCCAGAATC-3’ ，SEQ ID NO.24；

Ca-LF: 5’-CAAAGTAGTCAATACCTTCGAAC-3’ ，SEQ ID NO.25；

Ca-LR: 5’-TAGCTCCAGTAGAGAAGGTATT-3’ ，SEQ ID NO.26；

group 6, giardia specific primer (GD):

GD-F3: 5’-TCGTCGAGTGGATCCCG-3’ ，SEQ ID NO.27；

GD-B3: 5’- CCATCTCGTCCATCCCCTC -3’ ，SEQ ID NO.28；

GD-FIP3: 5’- GATGAAGGTCGCGGCCATCTTGAACAACATGAAGGTCAGCGT -3’ ，SEQ ID NO.29；

GD-BIP3: 5’- GCATCCAGGAGCTCTTCAAGCGTGTACCAGTGGAGGAAGGC -3’ ，SEQ ID NO.30；

group 7, primers specific to amoeba: (E. histolytica)：

Eh-F3: 5’- GCA CTA TAC TTGAAC GGA TTG -3’ ，SEQ ID NO.31；

Eh-B3: 5’- GTT TGA CAA GAT GTTGAG TGA -3’ ，SEQ ID NO.32；

Eh-FIP3: 5’-TCG CCC TAT ACT CAA ATA TGA CAA GAC TTT GGT

GGAAGA TTC ACG -3’ ，SEQ ID NO.33；

Eh-BIP3: 5’- ATC TAG TAG CTG GTTCCA CCT GAACAC CTA ATC ATT

ATC TTT ACC AAT C -3’ ，SEQ ID NO.34；

Group 8, primers specific for nosema peelii (EnW):

EnW-F3: 5’- TCGGAATGTRTDGTAGGTGA -3’ ，SEQ ID NO.35；

EnW -B3: 5’- CGACGGATCCAAGTGATC -3’ ，SEQ ID NO.36；

EnW -FIP3: 5’- TTGGTACGTGATGGTTGGATGGGCAACCTCCGATTTTCCT -3’ ，SEQ ID NO.37；

EnW -BIP3: 5’- GGAACCACCTCTAACTCACTGCTGTAGGCGTGAGAGTGTAT -3’ ，SEQ ID NO.38。

as a preferred technical scheme of the invention, the kit also comprises a LAMP reaction system as follows:

as the preferable technical scheme of the invention, the primer is freeze-dried and then solidified on the chip to prepare the honeycomb chip; the kit further comprises a color-developing agent, for example, calcein or the like.

In a second aspect of the present invention, a honeycomb chip-based PCR detection method for high-throughput detection of intestinal protozoa is provided, which comprises the following specific steps:

(1) DNA extraction of different worm strains, said worm strains comprising Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium turkey, Cryptosporidium andersoni, Giardia, amoeba, Microsporidium bicucrium bicolor, as defined in claim 1;

(2) designing and screening primers: the designed primer sequence is the primer sequence of claim 1;

(3) plasmid construction: adopting gene synthesis as a template, and constructing a plasmid as verification; each insect strain has a respective template;

(4) establishing a single-tube LAMP method, and carrying out LAMP detection on the designed primers to obtain effective amplification primers;

(5) performing amplification specificity verification of the gene chip according to the LAMP reaction condition in the step (4);

(6) and (3) detecting and verifying the feasibility of the sample according to the established nested PCR method.

As a preferred technical scheme of the invention, the step (3) is specifically as follows:

downloading the sequence of each insect strain according to a database of NCBI, comparing the sequences of different isolates of the same insect strain, and selecting a proper region for template synthesis; constructing a plasmid, and sequencing the plasmid to be consistent with a target sequence;

a total of 8 template plasmids:

(1) CRY-403bp, and the nucleotide sequence is shown as SEQ ID NO. 39;

(2) ch-244bp, and the nucleotide sequence is shown as SEQ ID NO. 40;

(3) cp-221bp, and the nucleotide sequence is shown as SEQ ID NO. 41;

(4) cm-408bp, and the nucleotide sequence is shown as SEQ ID NO. 42;

(5) ca-230bp, and the nucleotide sequence is shown as SEQ ID NO. 43;

(6) GD-810bp, the nucleotide sequence is shown as SEQ ID NO. 44;

(7) e.his-266bp, the nucleotide sequence is shown as SEQ ID NO. 45;

(8) EnW-310bp, and the nucleotide sequence is shown in SEQ ID NO. 46.

In a preferred embodiment of the present invention, in the step (4) and the step (5), the LAMP reaction conditions are:

mixing the above materials, placing in a constant temperature heating instrument at 60 deg.C for 50 min, and observing fluorescence (or verifying electrophoresis and fluorescence results).

In a third aspect of the invention, there is provided a primer set for detecting cryptosporidium, giardia, microsporidia and entamoeba histolytica, selected from any one or any combination of the following 8 sets of primers:

group 1, primers specific for Cryptosporidium (CRY):

CRY-F3: 5′-CTTACTCCTTCAGCACCTTA-3′，SEQ ID NO.1；

CRY -B3: 5′-CAAGAAAGAGCTATCAATCTGT-3′，SEQ ID NO.2；

CRY -BIP:5′-CCTGCGGCTTAATTTGACTCACAATCCTTCCTATGTCTGGAC-3′，SEQ ID NO.4；

CRY -LF: 5’-TGCGACCATACTCCCCCCA-3’，SEQ ID NO.5；

CRY -LB: 5’-ACACGGGAAAACTCACCAG-3’，SEQ ID NO.6；

group 2, primers specific for human cryptosporidium (Ch):

Ch-F3: 5’-GGCAATCAGGTTGAGTCA-3’ ，SEQ ID NO.7;

Ch-B3: 5’-CGGTATAGAAAGCACTATCGT-3’ ，SEQ ID NO.8;

Ch-FIP:5’-AGGCAAACAAATCGACGGTTG-AGATCAAGAAGATCACTCACA-3’ ，SEQ ID NO.9;

group 3, primers specific for Cryptosporidium parvum (Cp):

Cp-F3: 5’-TCGCAC CAGCAA ATA AGG C-3’ ，SEQ ID NO.11;

Cp-B3: 5’-GCCGCA TTC TTC TTT TGGAG-3’ ，SEQ ID NO.12;

Cp-FIP: 5’-ACCCTGGCTACCAGAAGCTTCAGAACTGGAGACGCAGAA-3’ ，SEQ ID NO.13;

Cp-BIP:5’-GGCCAAACTAGTGCTGCTTCCCGTTTCGGTAGTTGCGCCTT-3’ ，SEQ ID NO.14；

Cp-LF: 5’-GTACCACTAGAATCTTGACTGCC-3’ ，SEQ ID NO.15；

Cp-LB: 5’-AACCCACTACTCCAGCTCAAAGT-3’ ，SEQ ID NO.16；

group 4, primers specific for cryptosporidium turkey (Cm):

Cm-F3：5’-CCTTTGAAAACGAATCAAGTTCT-3’ ，SEQ ID NO.17;

Cm- B3：5’- CAATTCTTTTACCACCTTGGA-3’ ，SEQ ID NO.18;

group 5, primers specific for cryptosporidium andersi (Ca):

Ca-F3: 5’-CGTGCAAAGAGAACACTTTC-3’ ，SEQ ID NO.21；

Ca-B3: 5’-CCTACTAATACAACATCATGTACT-3’ ，SEQ ID NO.22；

Ca-LF: 5’-CAAAGTAGTCAATACCTTCGAAC-3’ ，SEQ ID NO.25；

Ca-LR: 5’-TAGCTCCAGTAGAGAAGGTATT-3’ ，SEQ ID NO.26；

group 6, giardia specific primer (GD):

GD-F3: 5’-TCGTCGAGTGGATCCCG-3’ ，SEQ ID NO.27；

GD-B3: 5’- CCATCTCGTCCATCCCCTC -3’ ，SEQ ID NO.28；

group 7, primers specific to amoeba: (E. histolytica)：

Eh-F3: 5’- GCA CTA TAC TTGAAC GGA TTG -3’ ，SEQ ID NO.31；

Eh-B3: 5’- GTT TGA CAA GAT GTTGAG TGA -3’ ，SEQ ID NO.32；

Eh-FIP3: 5’-TCG CCC TAT ACT CAA ATA TGA CAA GAC TTT GGT

GGAAGA TTC ACG -3’ ，SEQ ID NO.33；

Eh-BIP3: 5’- ATC TAG TAG CTG GTTCCA CCT GAACAC CTA ATC ATT

ATC TTT ACC AAT C -3’ ，SEQ ID NO.34；

Group 8, primers specific for nosema peelii (EnW):

EnW-F3: 5’- TCGGAATGTRTDGTAGGTGA -3’ ，SEQ ID NO.35；

EnW -B3: 5’- CGACGGATCCAAGTGATC -3’ ，SEQ ID NO.36；

in the fourth aspect of the invention, the application of the primer group in the preparation of a honeycomb chip-based kit for detecting the intestinal protozoa in a high-throughput manner is provided.

In the fifth aspect of the present invention, there is provided a method for detecting intestinal protozoan molecular biological nucleic acid, comprising the following steps:

(1) freeze-drying any group of primers, and then solidifying the primers on a chip to prepare an LAMP microfluidic chip;

(2) selecting a proper LAMP reaction system, adding a color developing agent, and adding the mixture into a chip;

(3) the reaction result can be judged by naked eyes or instruments.

As a preferential technical scheme of the invention, the step (1) is specifically as follows: assembling a plurality of capillaries into a customized base for fixing to form the LAMP microfluidic chip; then, selecting any one group of primers to dissolve in the primer fixing solution, respectively sucking the primer fixing solutions aiming at different targets, and adding the primer fixing solutions into corresponding capillary reaction holes on the capillary chip according to a pre-designed arrangement sequence; reserving two capillaries, wherein one capillary fixes a positive control primer group as a positive control hole, and the last capillary fixes a non-fixed primer group as a negative control hole; drying the capillary to fix the primer on the inner wall of the capillary.

As a preferential technical scheme of the invention, the step (2) is specifically that a sample to be detected, an LAMP reaction reagent and a color developing agent are added into a reaction hole of a microarray chip and reacted for 1 hour at 63 ℃.

As a preferential technical scheme of the invention, the reaction result detection and data analysis in the step (3) are specifically as follows: after the LAMP reaction is finished, the color development condition of all reaction holes on the chip is detected through ultraviolet fluorescence, and the detected result is judged to be positive or negative under the condition that the positive control hole develops color and the negative control hole does not develop color.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention relates to a nucleic acid high-throughput detection method based on a capillary microarray, which improves the detection throughput and detection efficiency, reduces the detection cost and sample consumption. The time and operability for detecting any insect species are superior to those of the conventional PCR technology, and the method is rapid and convenient; meanwhile, a high-end precise instrument is not needed, the operation is superior to that of fluorescent quantitative PCR, and the on-site rapid detection of intestinal protozoa is facilitated. In addition, the traditional LAMP technology is easy to cause aerosol false positive pollution in an operation space, and the method is based on the microfluidic platform, so that the pollution of the aerosol in the air can be reduced, and the incidence rate of the false positive can be reduced.

2. The honeycomb chip for detecting the intestinal protozoa, disclosed by the invention, is used for carrying out bioinformatics analysis on the 6 intestinal protozoa and designing primers capable of distinguishing respective insect species simultaneously, so that the time cost can be greatly saved, and the operation difficulty is reduced.

3. The primer has good specificity, and can distinguish different targets of intestinal protozoa.

4. The honeycomb chip detection method can be used for quickly and efficiently detecting the target in the actual sample, and is favorable for conveniently and quickly screening the sample infected by the intestinal protozoa.

Drawings

FIG. 1 is a diagram showing the results of verifying the specificity of the primers for Cryptosporidium in example 4 of the present invention. In FIG. 1, primers 1-9 are from the literature and tested, and corresponding actual samples 1-9 are used, and the detected samples are actual samples of Cryptosporidium bovis; 1 and 2 represent cryptosporidium hominis, 3 represents cryptosporidium andersoni, 4 represents cryptosporidium bovis, 5 and 6 represent giardia, 7 and 8 represent entamoeba histolytica, 9 represents microsporidian, and 10 represents cotton template as a positive control of the reaction system in a single-tube assay.

FIG. 2 is a diagram showing the results of verifying the specificity of primers for microsporidian in example 4 of the present invention. In fig. 2, 1 represents giardia's literature primer to detect the corresponding plasmid, 2 represents histolytica amoeba literature primer to detect the corresponding plasmid, 3 represents microsporidian primer to detect the corresponding plasmid by redesigning a primer, 4 represents newly designed cyclosporine primer to detect the corresponding plasmid, 5 represents literature-derived primer to detect the actual sample cryptosporidium andersoni, 6 represents literature-derived primer to detect the actual sample cryptosporidium parvum, 7-9 represents cryptosporidium universal primer to detect different actual samples, and 10 represents cotton template as a single-tube detected reaction system positive control.

FIG. 3 is a diagram showing the results of verifying the specificity of the Giardia primer in example 4 of the present invention. In FIG. 3, 1-5 represent primer verification for Giardia; 6-7 represent primer validation of histolytic amoeba; 8 represents the corresponding detection of corresponding plasmids by the primers in the cryptosporidium andersoni literature; 9 represents the corresponding detection of corresponding plasmid by using a cryptosporidium parvum literature primer; 10 represents cotton template as positive control of reaction system for single-tube detection.

FIG. 4 is a diagram showing the results of verifying the specificity of primers for Cryptosporidium hominis in example 4 of the present invention. In FIG. 4, 1 represents a cotton template as a positive control of a reaction system for single-tube detection; 2-3 represents the primer verification of the human cryptosporidium; 4-6 represent primer verification of Cryptosporidium turkey.

FIG. 5 is a schematic representation of the single tube validation results for eight parasite targets in example 4 of the present invention; wherein 1 represents a general primer of cryptosporidium, 2 represents a primer of cryptosporidium hominis, 3 represents a primer of cryptosporidium parvum, 4 represents a primer of cryptosporidium andersoni, 5 represents a primer of cryptosporidium turkey, 6 represents a primer of giardia, 7 represents a primer of amoeba histolytica, and 8 represents a primer of microsporidia.

FIG. 6 is a diagram showing the distribution of targets on a chip and the results of specificity verification in example 4 of the present invention.

FIG. 7 is a graph showing the results of the detection of the actual samples confirmed to contain the corresponding pathogens on the honeycomb chip in example 6 of the present invention.

Detailed Description

The following examples illustrate the invention in detail: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the present embodiment is only used for illustrating the present invention and is not used to limit the scope of the present invention. Experimental procedures in which specific conditions are not specified in the examples are generally carried out under conventional conditions, for example, as described in molecular cloning, A laboratory Manual (scientific Press, 1992), J.Sambrook et al, or as recommended by the manufacturer.

Experiment platform

The detection platform on which the invention is based is a honeycomb chip (platform) provided by Shanghai university of transportation, and the specific content of the detection platform is disclosed in the patent application with the patent application number of 201510901558.1 and the invention name of the invention is a high-throughput rapid detection method of nucleic acid based on capillary microarray. The honeycomb chip (platform) is processed into a microarray by means of capillary assembly, casting, machining and the like, a plurality of hydrophilic vertical microchannels are contained in the microarray, and the outer surface of the microarray is subjected to super-hydrophobic modification by a chemical method; adding a plurality of groups of nucleic acid amplification primers into a plurality of micro-pipelines respectively, drying and fixing, and placing the microarray into a reaction tube; then, introducing nucleic acid amplification reaction components into each micro-pipeline at one time by a special sample adding device in a siphon mode, and putting the components into a temperature control device for amplification reaction; real-time detection and end point detection are respectively realized by continuously measuring in the reaction or measuring the fluorescent signal in the micro-pipeline for one time after the reaction; the amplification product may also be recovered for subsequent use. The platform has the advantages that the detection of a plurality of nucleic acid targets can be quickly and conveniently realized in one reaction, and the platform is widely applied to the fields of nucleic acid multiplex detection, field detection and the like.

The honeycomb chip (platform) of the invention can simply realize the rapid parallel sample introduction of nucleic acid amplification reagents and the rapid parallel operation of amplification reaction in a micro system. Compared with the existing method, the method has the specific advantages of high detection flux, simple experiment operation, less sample consumption, low detection cost, no need of expensive equipment and the like. Can be widely applied in the fields of infectious disease rapid diagnosis, entry and exit inspection and quarantine, transgenic crop product field detection, food water source microorganism field detection, crime field evidence identification, biological counterterrorism and the like.

The specific detection method of the honeycomb chip (platform) comprises the following steps:

(1) adding a plurality of groups of nucleic acid amplification primers into a plurality of microchannels on a capillary microarray respectively, drying to facilitate the nucleic acid amplification primers to be attached to the inner walls of the microchannels, and fixing the capillary microarray in a transparent reaction tube;

(2) adding a nucleic acid amplification reaction component containing sample nucleic acid into a micro-pipeline to form a nucleic acid amplification system, and sealing a reaction pipe orifice;

(3) placing the reaction tube with the nucleic acid amplification system under a temperature-controlled condition for amplification reaction;

(4) real-time detection is realized by measuring continuous fluorescence signals of the amplification reaction or end-point detection is realized by measuring one-time fluorescence signals after the amplification reaction is finished.

Wherein, a plurality of micro-pipelines in the capillary micro-array penetrate and are arranged on the substrate in an array mode, and a small part of the end part of each micro-pipeline is exposed out of the surface of the substrate; the upper surface of the substrate, the outer surface of the micro-pipeline exposed out of the substrate part and the inner surface of the bottom end of the micro-pipeline are hydrophobic surfaces; the hydrophobic surface may be achieved by applying a layer of hydrophobic coating to the respective surface. The substrate is made of plastic, glass, metal and other high polymer materials; wherein, the other high molecular materials are polydimethylsiloxane, polymethyl methacrylate, polytetrafluoroethylene, rubber and the like; the micro-pipeline is a hydrophilic capillary, the part of the micro-pipeline exposed out of the substrate and the bottom end surface of the micro-pipeline are hydrophobic, and the micro-pipeline is made of hydrophilic materials. The step (1) is adding, in particular, a nucleic acid amplification primer is dissolved in a cross-linking agent and then added into a micro-pipeline; the cross-linking agent is any one of the following three mixed solutions:

a. 0.1-1% by mass of acetic acid aqueous solution of chitosan, and the pH value is 4.5-6.0;

b. gelatin water solution with the mass percent of 0.1-1%;

c. 0.05-5% of polyethylene glycol aqueous solution by mass percent.

The reaction component added in the step (1) is specifically introduced into each micro-pipeline in an inverted siphon mode. The inverted siphon mode is as follows: the sample cell of the sample adding device filled with the nucleic acid amplification reaction component solution is inserted into the reaction tube downwards, the nucleic acid amplification reaction component is contacted with the top end of the inner wall of the hydrophilic micro-tube and then quickly filled in the hydrophilic tube under the action of siphon, and then the sample adding device is removed. The measurement in (4) can be performed by means of fluorescence detection equipment or photometric detection equipment, or can be performed by visually distinguishing color or brightness differences, namely, before each measurement, a light source with corresponding emission wavelength is required to irradiate the reactant in the microchannel, and then the measurement is performed. In the method, the temperature control device in (3) and the measurement in (4) can be integrated into an automatic device, and the automatic operation of the automatic device is controlled by a software program. The method can be realized in a single reaction tube, or can be realized in parallel in an integrated 8-tube, 96-well plate or 384-well plate.

The platform has the advantages that: (1) by utilizing the capillary microarray and the hydrophilic and hydrophobic characteristics, the reaction liquid can be quickly and conveniently added into a plurality of microchannels at one time by adopting a specially designed sample adding device, so that the flux and the detection efficiency of one-time detection are improved; (2) the micro-pipeline is used as the reaction cavity, so that the dosage of a reagent sample is greatly reduced, and the detection cost is reduced; (3) the method is suitable for the fields of high-flux rapid detection of various nucleic acids, such as rapid detection of infectious diseases, entry and exit inspection and quarantine, food safety and transgenic detection, criminal investigation identification and the like.

Example 1 DNA extraction of different insect strains.

(1) The positive samples of cryptosporidium, giardia, amoeba histolytica and microsporidia are obtained by laboratory separation, potassium dichromate is stored at 4 ℃.

(2) The field samples are taken from diarrhea patients and animal waste samples in hospitals of Shanghai, Jiangsu and the like.

Feces DNA extraction kit was purchased from Qiagen, USA, and PCR mix was purchased from Bio-Server.

II, carrying out molecular detection on related pathogens by the nucleic acid of the sample according to the technical scheme for detecting diarrhea syndrome, and verifying the insect species of the sample through sequencing and comparison.

Example 25 detection primers for intestinal protozoa autonomous design and screening

(1) According to bioinformatics analysis, different target genes are respectively adopted for target design.

(2) And (3) designing a primer.

(3) Primer screening

Screening strategy: performing literature retrieval on each insect strain, selecting a literature report primer for verification, and if the primer is effective, taking the primer as a spare primer of the chip; if the primers can not be specifically amplified, a plurality of sequences are compared according to the sequences of the literature, and possible primers are designed to be verified one by one, so that effective specific amplification primers are obtained.

Wherein the sequences of the cryptosporidium hominis, the cryptosporidium turkey, the giardia and the amoeba are independently designed and synthesized; the general primers of cryptosporidium (2 loop primers are associated), cryptosporidium parvum and cryptosporidium andersoni adopt literature primers; 1 primer of microsporidian is designed as degenerate primer. The primers are specifically as follows:

group 1, primers specific for Cryptosporidium (CRY):

CRY-F3: 5′-CTTACTCCTTCAGCACCTTA-3′，SEQ ID NO.1；

CRY -B3: 5′-CAAGAAAGAGCTATCAATCTGT-3′，SEQ ID NO.2；

CRY -BIP:5′-CCTGCGGCTTAATTTGACTCACAATCCTTCCTATGTCTGGAC-3′，SEQ ID NO.4；

CRY -LF: 5’-TGCGACCATACTCCCCCCA-3’，SEQ ID NO.5；

CRY -LB: 5’-ACACGGGAAAACTCACCAG-3’，SEQ ID NO.6；

group 2, primers specific for human cryptosporidium (Ch):

Ch-F3: 5’-GGCAATCAGGTTGAGTCA-3’ ，SEQ ID NO.7;

Ch-B3: 5’-CGGTATAGAAAGCACTATCGT-3’ ，SEQ ID NO.8;

Ch-FIP:5’-AGGCAAACAAATCGACGGTTG-AGATCAAGAAGATCACTCACA-3’ ，SEQ ID NO.9;

group 3, primers specific for Cryptosporidium parvum (Cp):

Cp-F3: 5’-TCGCAC CAGCAA ATA AGG C-3’ ，SEQ ID NO.11;

Cp-B3: 5’-GCCGCA TTC TTC TTT TGGAG-3’ ，SEQ ID NO.12;

Cp-FIP: 5’-ACCCTGGCTACCAGAAGCTTCAGAACTGGAGACGCAGAA-3’ ，SEQ ID NO.13;

Cp-BIP:5’-GGCCAAACTAGTGCTGCTTCCCGTTTCGGTAGTTGCGCCTT-3’ ，SEQ ID NO.14；

Cp-LF: 5’-GTACCACTAGAATCTTGACTGCC-3’ ，SEQ ID NO.15；

Cp-LB: 5’-AACCCACTACTCCAGCTCAAAGT-3’ ，SEQ ID NO.16；

group 4, primers specific for cryptosporidium turkey (Cm):

Cm-F3：5’-CCTTTGAAAACGAATCAAGTTCT-3’ ，SEQ ID NO.17;

Cm- B3：5’- CAATTCTTTTACCACCTTGGA-3’ ，SEQ ID NO.18;

group 5, primers specific for cryptosporidium andersi (Ca):

Ca-F3: 5’-CGTGCAAAGAGAACACTTTC-3’ ，SEQ ID NO.21；

Ca-B3: 5’-CCTACTAATACAACATCATGTACT-3’ ，SEQ ID NO.22；

Ca-FIP: 5’-TCCTCAAATCTTGCACGACTTATWGATTCATCTACWC

AAGCAACAAT-3’ ，SEQ ID NO.23；

Ca-LF: 5’-CAAAGTAGTCAATACCTTCGAAC-3’ ，SEQ ID NO.25；

Ca-LR: 5’-TAGCTCCAGTAGAGAAGGTATT-3’ ，SEQ ID NO.26；

group 6, giardia specific primer (GD):

GD-F3: 5’-TCGTCGAGTGGATCCCG-3’ ，SEQ ID NO.27；

GD-B3: 5’- CCATCTCGTCCATCCCCTC -3’ ，SEQ ID NO.28；

group 7, primers specific to amoeba: (E. histolytica)：

Eh-F3: 5’- GCA CTA TAC TTGAAC GGA TTG -3’ ，SEQ ID NO.31；

Eh-B3: 5’- GTT TGA CAA GAT GTTGAG TGA -3’ ，SEQ ID NO.32；

Eh-FIP3: 5’-TCG CCC TAT ACT CAA ATA TGA CAA GAC TTT GGT

GGAAGA TTC ACG -3’ ，SEQ ID NO.33；

Eh-BIP3: 5’- ATC TAG TAG CTG GTTCCA CCT GAACAC CTA ATC ATT

ATC TTT ACC AAT C -3’ ，SEQ ID NO.34；

Group 8, primers specific for nosema peelii (EnW):

EnW-F3: 5’- TCGGAATGTRTDGTAGGTGA -3’ ，SEQ ID NO.35；

EnW -B3: 5’- CGACGGATCCAAGTGATC -3’ ，SEQ ID NO.36；

example 3 plasmid construction

a total of 8 template plasmids:

(1) CRY-403bp, and the nucleotide sequence is shown as SEQ ID NO. 39;

(2) ch-244bp, and the nucleotide sequence is shown as SEQ ID NO. 40;

(3) cp-221bp, and the nucleotide sequence is shown as SEQ ID NO. 41;

(4) cm-408bp, and the nucleotide sequence is shown as SEQ ID NO. 42;

(5) ca-230bp, and the nucleotide sequence is shown as SEQ ID NO. 43;

(6) GD-810bp, the nucleotide sequence is shown as SEQ ID NO. 44;

(7) e.his-266bp, the nucleotide sequence is shown as SEQ ID NO. 45;

(8) EnW-310bp, and the nucleotide sequence is shown in SEQ ID NO. 46.

Example 4 validation of primer effectiveness by Single tube LAMP

Specific experimental steps for verifying primer effectiveness of single tube LAMP

(1) Adding the nucleic acid amplification primers of any insect species into a 0.2ml PCR tube respectively, wherein the system is as follows:

(2) adding 1 mu l of nucleic acid amplification reaction components containing sample nucleic acid into a 0.2ml PCR tube to form a complete nucleic acid amplification system, and sealing the mouth of the reaction tube;

(5) And (3) carrying out electrophoresis verification on reaction products in the reaction tube, carrying out comparative electrophoresis on each sample by adopting negative control and positive reaction, and presenting a characteristic DNA fragment image with different sizes, namely positive amplification.

Second, single tube LAMP and honeycomb chip verification primer specificity

1. The experimental method comprises the following steps: the specific method for optimizing the PCR reaction conditions comprises the following steps: LAMP reaction system

The LAMP reaction system adopted is as follows:

after mixing the above, the mixture was left at 60 ℃ for 1 hour in a PCR instrument, and then fluorescence was observed, and electrophoresis was carried out.

As a result: the primers of the above 8 insect species are all effective, and the primer marked red is self-original.

2. The experimental results are as follows:

(1) CRY can effectively amplify the bovine cryptosporidium.

As shown in FIG. 1, S4 is a primer for Cryptosporidium (CRY), and the primer is verified by detecting the real sample of bovine Cryptosporidium. In FIG. 1, primers 1-9 were from the literature and tested, 1-9 using the corresponding actual samples, 1 and 2 for Cryptosporidium hominis, 3 for Cryptosporidium andersoni, 4 for Cryptosporidium bovis, 5 and 6 for Giardia, 7 and 8 for Entamoeba histolytica, 9 for Microsporidium parvum, and 10 for Cotton template as a positive control in the reaction system for single-tube assay. The specificity of the Cryptosporidium (CRY) primer can be confirmed from the results of FIG. 1.

(2) Microsporidium parvum (EnW) primers and efficient amplification of cryptosporidium andersoni and cryptosporidium parvum

As shown in FIG. 2, after the primers of microsporidian are analyzed for biological information, one primer is updated, and the detection of plasmid is verified; meanwhile, the literature primers of cryptosporidium andersoni and cryptosporidium parvum can effectively detect the actual sample; the universal primers for Cryptosporidium can detect Cryptosporidium in 3 actual samples.

In fig. 2, 1 represents giardia's literature primer to detect the corresponding plasmid, 2 represents ameba-lysing literature primer to detect the corresponding plasmid, 3 represents redesigned microsporidian's primer FIB and examination of the corresponding plasmid, 4 represents newly designed circumsporozoite primer to detect the corresponding plasmid, 5 represents literature-derived primer to detect cryptosporidium andersoni in the actual sample, 6 represents literature-derived primer to detect cryptosporidium parvum in the actual sample, 7-9 represents cryptosporidium universal primer to detect different actual samples, and 10 represents cotton template as a positive control of a single-tube detection reaction system.

(3) Effective design primers of giardia and ameba histolytica and effective detection of corresponding plasmids of cryptosporidium andersoni and cryptosporidium parvum primers

FIG. 3 demonstrates primers designed for 5 sets of Giardia, one set of which can efficiently detect the corresponding plasmid; 2 groups of designed primers of tissue-dissolving amoeba prove that one group is effective;

in FIG. 3, 1-5 represent primer verification for Giardia; 6-7 represent primer validation of histolytic amoeba; 8 represents the corresponding detection of corresponding plasmids by the primers in the cryptosporidium andersoni literature; 9 represents the corresponding detection of corresponding plasmid by using a cryptosporidium parvum literature primer; 10 represents cotton template as positive control of reaction system for single-tube detection.

(4) Design of human Cryptosporidium (A), (B), (C)C. hominis) And verification of Cryptosporidium turkey (C. meleagris) primers

As shown in fig. 4, 2 sets of primers of cryptosporidium hominis were designed, wherein one set of primers was effective in detecting the corresponding plasmid; the primers of 3 groups of cryptosporidium turkey were designed to be effective.

In FIG. 4, 1 represents a cotton template as a positive control of a reaction system for single-tube detection; 2-3 represents the primer verification of the human cryptosporidium; 4-6 represent primer verification of Cryptosporidium turkey.

(5) All 8 parasite primers were efficiently amplified

Results are shown in table 1 and fig. 5, a single tube validation results schematic for eight parasite targets in example 4 of the invention is listed in table 1 (fig. 5); wherein 1 represents a general primer of cryptosporidium, 2 represents a primer of cryptosporidium hominis, 3 represents a primer of cryptosporidium parvum, 4 represents a primer of cryptosporidium andersoni, 5 represents a primer of cryptosporidium turkey, 6 represents a primer of giardia, 7 represents a primer of amoeba histolytica, and 8 represents a primer of microsporidia. The specific primers can effectively detect the corresponding plasmids.

TABLE 1

(6) Verification of amplification specificity of Gene chip according to the LAMP reaction conditions

Chip composition 6 targets were placed as in figure 6; chip reactions were performed on the chip using the respective plasmids to verify primer specificity. FIG. 6 is a diagram showing the distribution of targets on a chip and the results of specificity verification in example 4 of the present invention. 6 specific insect species primers are anchored in a microfluid honeycomb chip for detection, and the result proves that each primer has specificity.

Example 5 detection with real samples

The field samples are taken from diarrhea patients and animal waste samples in hospitals of Shanghai, Jiangsu and the like. Fecal DNA extraction kits were purchased from QIAamp DNA fecal extraction kits from Qiagen, Germany, and stored at-20 ℃ according to the kit instructions. PCR mix was purchased from Bio-Server.

Samples identification of pathogens infected in actual samples was performed using nested PCR according to the reaction system of table 3 and the reaction procedure of table 4, following the primers (table 2) involved in the diarrhea syndrome monitoring protocol (2019 version).

Table 2: nested PCR primer verification of actual samples

The PCR reaction systems for the four protozoa are shown in Table 3, and the reaction procedures are shown in Table 4.

Table 3: single gene PCR amplification reaction system

Table 4: nested PCR cycle parameters

And (3) identifying a PCR product: 5. mu.l of the PCR product was electrophoretically analyzed on a 2% agarose gel (120V/40 min).

Note that: each sample was amplified at least 3 times by parallel PCR, each time with positive and negative controls. The obtained positive detection sample is sequenced and verified to be cryptosporidium, giardia, microsporidian and entamoeba histolytica.

Example 6A method for detecting intestinal protozoan molecular biological nucleic acid

The method comprises the following steps:

(1) manufacturing and processing an LAMP microfluidic chip: assembling 10 hydrophobized capillaries into a customized base, and fixing for later use;

(2) and (3) primer fixation: respectively selecting any one group of primers in the embodiment 2 to be dissolved in the primer fixing solution, respectively sucking the primer fixing solutions aiming at different targets, and adding the primer fixing solutions into corresponding capillary reaction holes on the capillary chip according to a pre-designed arrangement sequence; two capillaries are reserved, wherein one capillary fixes a positive control primer group as a positive control hole, and the last capillary fixes a non-fixed primer group as a negative control hole. Drying the capillary tube to fix the primer on the inner wall of the capillary tube;

(3) LAMP reaction on a chip: adding a sample to be detected, an LAMP reaction reagent and calcein into a reaction hole of the microarray chip through an auxiliary sample adding device, and reacting for 1 hour at 63 ℃;

(4) and (3) result detection and data analysis: after the LAMP reaction is finished, the color development condition of all reaction holes on the chip is detected through ultraviolet fluorescence, and the detected result is judged to be positive or negative under the condition that the positive control hole develops color and the negative control hole does not develop color.

(5) FIG. 7 is a graph showing the results of the detection of the present invention using the actual samples confirmed to contain the corresponding pathogens in example 5 for the honeycomb chip. As shown in FIG. 7, the samples of human Cryptosporidium Anseri, Cryptosporidium turkey, Giardia, amoeba histolytica, and Microsporidium were used for verification, respectively, to obtain accurate verification. The arrangement is as per fig. 7.

Sequence listing

<110> Chinese disease prevention and control center for prevention and control of parasitic diseases institute (national center for research on tropical diseases)

<120> honeycomb chip-based kit for high-throughput detection of intestinal protozoa and detection method

<130> WH-NP-20-100609

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aatgcgaaag catttgccaa ggatgttttc attaatcaag aacgaaagtt aggggatcga 60

agacgatcag ataccgtcgt agtcttaacc ataaactatg ccaactagag attggaggtt 120

gttccttact ccttcagcac cttatgagaa atcaaagtct ttgggttctg gggggagtat 180

ggtcgcaagg ctgaaactta aaggaattga cggaagggca ccaccaggag tggagcctgc 240

ggcttaattt gactcaacac gggaaaactc accaggtcca gacataggaa ggattgacag 300

attgatagct ctttcttgat tctatgggtg gtggtgcatg gccgttctta gttggtggag 360

tgatttgtct ggttaattcc gttaacgaac gagaccttaa cct 403

<210> 40

<211> 244

<212> DNA

<213> Artificial sequence (unknown)

<400> 40

tctgctagct caagtagtcc aaccgaaaat aaaggtgagt ctggcaatca ggttgagtca 60

agatcaagaa gatcactcac agaggaaact agtgaaactg caaccgtcga tttgtttgcc 120

tttaccctta atggtggtaa gagaattgaa gtggctgtgc caaacgccga agaaacatcg 180

aaaagagaca agtacagttt ggttgcagac gatagtgctt tctataccgg caaaaatagc 240

ggca 244

<210> 41

<211> 221

<212> DNA

<213> Artificial sequence (unknown)

<400> 41

tcaaccgtcg caccagcaaa taaggcaaga actggagaag acgcagaagg cagtcaagat 60

tctagtggta ctgaagcttc tggtagccag ggttctgaag aggaaggtag tgaagacgat 120

ggccaaacta gtgctgcttc ccaacccact actccagctc aaagtgaagg cgcaactacc 180

gaaaccatag aagctactcc aaaagaagaa tgcggcactt c 221

<210> 42

<211> 408

<212> DNA

<213> Artificial sequence (unknown)

<400> 42

gtggatctac taatccaaat gaatgtggta cttcatttgt aatgtggttc caacatggca 60

ccccagttgc gactctgaag tgtggtgatt acactatcgt ttatgcacct gaaagtgaca 120

aaacagatcc cgctccaaaa tatatctctg gtgacgttaa ggctgtaacc tttgaaaacg 180

aatcaagttc taatacaata aaaatcaagg ttgacggtat ggagctcagc actctctcta 240

ctaattcaaa ttccccaaca gaaaatgaca ctgcgtctga ggaaagcttg tctagatcgc 300

gatcaaaaag atcactcaca gacgctgaga caactggaac tgttgatgtg cttgccttta 360

ccctccaagg tggtaaaaga attgaagtgg ctataccaaa tgccagtg 408

<210> 43

<211> 230

<212> DNA

<213> Artificial sequence (unknown)

<400> 43

agtgtgaacg tgcaaagaga acactttctt catctacaca agcaacaatc gaattggatt 60

ctttgttcga aggtattgac tactttgtat ctataagtcg tgcaagattt gaggaacttt 120

gttctgatta tttccgtggc acattagctc cagtagagaa ggtattaaaa gattctggaa 180

tggataagag gtcagtacat gatgttgtat tagtaggtgg ttcaacccgt 230

<210> 44

<211> 810

<212> DNA

<213> Artificial sequence (unknown)

<400> 44

ggcggcacgg gcgccgggat gggcacgctc ctcatcgcga agatccgcga ggagtacccc 60

gaccgcatga tgtgcacgtt ctccgtcgtc ccgtccccga aggtctcgga cacggtcgtt 120

gagccgtaca acgcgaccct ctcggtccac cagctcgtcg agcacgccga cgaggtcttc 180

tgcatcgaca acgaggccct ctacgacatc tgcttccgca cgctcaagct cacgtgcccc 240

acctacggag acctcaacca cctcgtctcg ctcgtcatgt ccggctgcac gagctgcctc 300

cgcttccccg gccagctcaa cgccgacctc cgcaagctcg cggtcaacct gatcccgttc 360

ccgcgcctcc acttcttcct cgtcggcttc gcgcccctga cgagccgcgg ctcccagatc 420

taccgcgccc tcacggtccc cgagctcgtc tcccagatgt tcgacaacaa gaacatgatg 480

gccgcgtccg acccgcgcca cggccgctac ctgaccgccg ccgccatgtt ccgcgggcgc 540

atgtccacga aggaggtcga cgagcagatg ctcaacatcc agaacaagaa ctcctcgtac 600

ttcgtcgagt ggatcccgaa caacatgaag gtcagcgtct gcgacatccc gccgcgcggg 660

ctcaagatgg ccgcgacctt catcggaaac tccacgtgca tccaggagct cttcaagcgc 720

gtcggcgagc agttcacggc gatgttccgc cgcaaggcct tcctccactg gtacacgggc 780

gaggggatgg acgagatgga gttcacagag 810

<210> 45

<211> 266

<212> DNA

<213> Artificial sequence (unknown)

<400> 45

aaagatggtg cactatactt gaacggattg aagccgaagg aaactttggt ggaagattca 60

cgcagtgtta acgtgcaaat tacttgtcat atttgagtat agggcgaaag actcatcgaa 120

ctatctagta gctggttcca cctgaatatt cccttaggaa ggtcaaagta ttaatagtat 180

tgattggtaa agataatgat taggtgtaat gggtgagtat attcactcaa catcttgtca 240

aactatgaat aaacaaagaa gaattc 266

<210> 46

<211> 310

<212> DNA

<213> Artificial sequence (unknown)

<400> 46

gtgggtatcg gaatgtatgg taggtgatgt gtgtgtgtat gggggatgcc gaggggacca 60

gcagtgcggt ggtgtgtgta ggcgtgagag tgtatctgca agggtgaggg atgtgggtgc 120

agtgagttag aggtggttcc atgtggaata gtgggattgg tacgtgatgg ttggatgggg 180

gaatgatgtg tgtatgggtg aggaaaatcg gaggttgcgg tgcgagcggc agtagggtgc 240

catcaagagg tgtatttgga aatatcccta atacaggatc acttggatcc gtcggcgatg 300

acgagcgcga 310

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 47

ttctagagct aatacatgcg 20

<210> 48

<211> 21

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 48

cccatttcct tcgaaacagg a 21

<210> 49

<211> 26

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 49

ggaagggttg tatttattag ataaag 26

<210> 50

<211> 22

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 50

aaggagtaag gaacaacctc ca 22

<210> 51

<211> 19

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 51

aaatiaigcc tgctcgtcg 19

<210> 52

<211> 19

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 52

caaaccttit ccgcaaacc 19

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 53

cccttcatcg giggtaactt 20

<210> 54

<211> 20

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 54

gtggccacca cicccgigcc 20

<210> 55

<211> 20

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 55

ttcagatggt catagggatg 20

<210> 56

<211> 18

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 56

attagagcat tccgtgag 18

<210> 57

<211> 20

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 57

tcggctctga atatctatgg 20

<210> 58

<211> 17

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 58

attctttcgc gctcgtc 17

<210> 59

<211> 18

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 59

tgctgtgatt aaaacgct 18

<210> 60

<211> 19

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 60

ttaactattt caatctcgg 19

<210> 61

<211> 25

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 61

acattttgaa gactttatgt aagta 25

<210> 62

<211> 24

<212> DNA

<213> Artificial sequence (unknown)

<220>

<221> misc_feature

<223> primer

<400> 62

cagatctaga aacaatgctt ctct 24

Claims

1. A kit for detecting intestinal protozoa in high flux based on a honeycomb chip is characterized in that a microfluidic system is integrated on the honeycomb chip and is used for detecting cryptosporidium, giardia, microsporidian and entamoeba histolytica, and the kit comprises any one or any combination of more than 8 groups of primers as follows:

group 1, primers specific for Cryptosporidium (CRY):

CRY-F3: 5′-CTTACTCCTTCAGCACCTTA-3′，SEQ ID NO.1；

CRY -B3: 5′-CAAGAAAGAGCTATCAATCTGT-3′，SEQ ID NO.2；

CRY -BIP:5′-CCTGCGGCTTAATTTGACTCACAATCCTTCCTATGTCTGGAC-3′，SEQ ID NO.4；

CRY -LF: 5’-TGCGACCATACTCCCCCCA-3’，SEQ ID NO.5；

CRY -LB: 5’-ACACGGGAAAACTCACCAG-3’，SEQ ID NO.6；

group 2, primers specific for human cryptosporidium (Ch):

Ch-F3: 5’-GGCAATCAGGTTGAGTCA-3’ ，SEQ ID NO.7;

Ch-B3: 5’-CGGTATAGAAAGCACTATCGT-3’ ，SEQ ID NO.8;

Ch-FIP:5’-AGGCAAACAAATCGACGGTTG-AGATCAAGAAGATCACTCACA-3’ ，SEQ ID NO.9;

group 3, primers specific for Cryptosporidium parvum (Cp):

Cp-F3: 5’-TCGCAC CAGCAA ATA AGG C-3’ ，SEQ ID NO.11;

Cp-B3: 5’-GCCGCA TTC TTC TTT TGGAG-3’ ，SEQ ID NO.12;

Cp-FIP: 5’-ACCCTGGCTACCAGAAGCTTCAGAACTGGAGACGCAGAA-3’ ，SEQ ID NO.13;

Cp-BIP:5’-GGCCAAACTAGTGCTGCTTCCCGTTTCGGTAGTTGCGCCTT-3’ ，SEQ ID NO.14；

Cp-LF: 5’-GTACCACTAGAATCTTGACTGCC-3’ ，SEQ ID NO.15；

Cp-LB: 5’-AACCCACTACTCCAGCTCAAAGT-3’ ，SEQ ID NO.16；

group 4, primers specific for cryptosporidium turkey (Cm):

Cm-F3：5’-CCTTTGAAAACGAATCAAGTTCT-3’ ，SEQ ID NO.17;

Cm- B3：5’- CAATTCTTTTACCACCTTGGA-3’ ，SEQ ID NO.18;

group 5, primers specific for cryptosporidium andersi (Ca):

Ca-F3: 5’-CGTGCAAAGAGAACACTTTC-3’ ，SEQ ID NO.21；

Ca-B3: 5’-CCTACTAATACAACATCATGTACT-3’ ，SEQ ID NO.22；

Ca-LF: 5’-CAAAGTAGTCAATACCTTCGAAC-3’ ，SEQ ID NO.25；

Ca-LR: 5’-TAGCTCCAGTAGAGAAGGTATT-3’ ，SEQ ID NO.26；

group 6, giardia specific primer (GD):

GD-F3: 5’-TCGTCGAGTGGATCCCG-3’ ，SEQ ID NO.27；

GD-B3: 5’- CCATCTCGTCCATCCCCTC -3’ ，SEQ ID NO.28；

group 7, primers specific to amoeba: (E. histolytica) ：

Eh-F3: 5’- GCA CTA TAC TTGAAC GGA TTG -3’ ，SEQ ID NO.31；

Eh-B3: 5’- GTT TGA CAA GAT GTTGAG TGA -3’ ，SEQ ID NO.32；

Eh-FIP3: 5’-TCG CCC TAT ACT CAA ATA TGA CAA GAC TTT GGT GGAAGA TTC ACG -3’ ，SEQ ID NO.33；

Eh-BIP3: 5’- ATC TAG TAG CTG GTTCCA CCT GAACAC CTA ATC ATT

ATC TTT ACC AAT C -3’ ，SEQ ID NO.34；

Group 8, primers specific for nosema peelii (EnW):

EnW-F3: 5’- TCGGAATGTRTDGTAGGTGA -3’ ，SEQ ID NO.35；

EnW -B3: 5’- CGACGGATCCAAGTGATC -3’ ，SEQ ID NO.36；

2. the kit of claim 1, further comprising a LAMP reaction system as follows:

double distilled water 5.1μl 100Mm MgSO₄ 2μl 10 Thermo buffer 2.5 μl 2.5mM dNTP 1.4μl 1.25mM Calcein 0.5μl 25mM MnCl₂ 0.5μl 5M Betaine 4μl 8000 U/Ml Bst 1μl 20 μ M of any one or more of the 8 sets of primers of claim 1 3.5μl Template DNA 1μl

。

3. The kit of claim 1, wherein the primers are lyophilized and cured onto the chip to form a honeycomb chip; the kit also comprises a color developing agent.

4. A PCR detection method for detecting intestinal protozoa in high flux based on a honeycomb chip is characterized by comprising the following specific steps:

5. The method according to claim 4, wherein step (3) is specifically:

a total of 8 template plasmids:

(1) CRY-403bp, and the nucleotide sequence is shown as SEQ ID NO. 39;

(2) ch-244bp, and the nucleotide sequence is shown as SEQ ID NO. 40;

(3) cp-221bp, and the nucleotide sequence is shown as SEQ ID NO. 41;

(4) cm-408bp, and the nucleotide sequence is shown as SEQ ID NO. 42;

(5) ca-230bp, and the nucleotide sequence is shown as SEQ ID NO. 43;

(6) GD-810bp, the nucleotide sequence is shown as SEQ ID NO. 44;

(7) e.his-266bp, the nucleotide sequence is shown as SEQ ID NO. 45;

(8) EnW-310bp, and the nucleotide sequence is shown in SEQ ID NO. 46.

6. The method of claim 4, wherein in step (4) and step (5), the LAMP reaction conditions are:

5.1. mu.l of double distilled water

100Mm MgSO4 2μl

10X Thermo buffer 2.5. mu.l

2.5mM dNTP 1.4μl

1.25mM Calcein 0.5μl

25mM MnCl2 0.5μl

5M Betaine 4μl

8000 U/Ml Bst 1μl

20 μ M3.5 μ l of any one or more of the 8 sets of primers of claim 1

DNA 1μl

Mixing the above materials, placing in a constant temperature heating instrument at 60 deg.C for 50 min, and observing fluorescence or verifying electrophoresis and fluorescence results.

7. The primer group for detecting the cryptosporidium, giardia, microsporidian and soluble tissue amoeba is characterized in that any one or any combination of more than 8 primer groups are selected from the following group:

group 1, primers specific for Cryptosporidium (CRY):

CRY-F3: 5′-CTTACTCCTTCAGCACCTTA-3′，SEQ ID NO.1；

CRY -B3: 5′-CAAGAAAGAGCTATCAATCTGT-3′，SEQ ID NO.2；

CRY -BIP:5′-CCTGCGGCTTAATTTGACTCACAATCCTTCCTATGTCTGGAC-3′，SEQ ID NO.4；

CRY -LF: 5’-TGCGACCATACTCCCCCCA-3’，SEQ ID NO.5；

CRY -LB: 5’-ACACGGGAAAACTCACCAG-3’，SEQ ID NO.6；

group 2, primers specific for human cryptosporidium (Ch):

Ch-F3: 5’-GGCAATCAGGTTGAGTCA-3’ ，SEQ ID NO.7;

Ch-B3: 5’-CGGTATAGAAAGCACTATCGT-3’ ，SEQ ID NO.8;

Ch-FIP:5’-AGGCAAACAAATCGACGGTTG-AGATCAAGAAGATCACTCACA-3’ ，SEQ ID NO.9;

group 3, primers specific for Cryptosporidium parvum (Cp):

Cp-F3: 5’-TCGCAC CAGCAA ATA AGG C-3’ ，SEQ ID NO.11;

Cp-B3: 5’-GCCGCA TTC TTC TTT TGGAG-3’ ，SEQ ID NO.12;

Cp-FIP: 5’-ACCCTGGCTACCAGAAGCTTCAGAACTGGAGACGCAGAA-3’ ，SEQ ID NO.13;

Cp-BIP:5’-GGCCAAACTAGTGCTGCTTCCCGTTTCGGTAGTTGCGCCTT-3’ ，SEQ ID NO.14；

Cp-LF: 5’-GTACCACTAGAATCTTGACTGCC-3’ ，SEQ ID NO.15；

Cp-LB: 5’-AACCCACTACTCCAGCTCAAAGT-3’ ，SEQ ID NO.16；

group 4, primers specific for cryptosporidium turkey (Cm):

Cm-F3：5’-CCTTTGAAAACGAATCAAGTTCT-3’ ，SEQ ID NO.17;

Cm- B3：5’- CAATTCTTTTACCACCTTGGA-3’ ，SEQ ID NO.18;

group 5, primers specific for cryptosporidium andersi (Ca):

Ca-F3: 5’-CGTGCAAAGAGAACACTTTC-3’ ，SEQ ID NO.21；

Ca-B3: 5’-CCTACTAATACAACATCATGTACT-3’ ，SEQ ID NO.22；

Ca-LF: 5’-CAAAGTAGTCAATACCTTCGAAC-3’ ，SEQ ID NO.25；

Ca-LR: 5’-TAGCTCCAGTAGAGAAGGTATT-3’ ，SEQ ID NO.26；

group 6, giardia specific primer (GD):

GD-F3: 5’-TCGTCGAGTGGATCCCG-3’ ，SEQ ID NO.27；

GD-B3: 5’- CCATCTCGTCCATCCCCTC -3’ ，SEQ ID NO.28；

group 7, primers specific to amoeba: (E. histolytica) ：

Eh-F3: 5’- GCA CTA TAC TTGAAC GGA TTG -3’ ，SEQ ID NO.31；

Eh-B3: 5’- GTT TGA CAA GAT GTTGAG TGA -3’ ，SEQ ID NO.32；

Eh-FIP3: 5’-TCG CCC TAT ACT CAA ATA TGA CAA GAC TTT GGT

GGAAGA TTC ACG -3’ ，SEQ ID NO.33；

Eh-BIP3: 5’- ATC TAG TAG CTG GTTCCA CCT GAACAC CTA ATC ATT

ATC TTT ACC AAT C -3’ ，SEQ ID NO.34；

Group 8, primers specific for nosema peelii (EnW):

EnW-F3: 5’- TCGGAATGTRTDGTAGGTGA -3’ ，SEQ ID NO.35；

EnW -B3: 5’- CGACGGATCCAAGTGATC -3’ ，SEQ ID NO.36；

8. use of the primer set according to claim 7 for preparing a honeycomb chip-based kit for high-throughput detection of intestinal protozoa.

9. A method for detecting intestinal protozoan molecular biological nucleic acid is characterized by comprising the following steps:

(1) freeze-drying any one group of primers of claim 7, and then solidifying the primers on a chip to prepare an LAMP microfluidic chip;

(3) the reaction result can be judged by naked eyes or instruments.

10. The method according to claim 9, wherein step (1) is specifically: assembling a plurality of capillaries into a customized base for fixing to form the LAMP microfluidic chip; then, selecting any one group of primers in claim 7 to be dissolved in the primer fixing solution, respectively sucking the primer fixing solutions aiming at different targets, and adding the primer fixing solutions into corresponding capillary reaction holes on the capillary chip according to a pre-designed arrangement sequence; reserving two capillaries, wherein one capillary fixes a positive control primer group as a positive control hole, and the last capillary fixes a non-fixed primer group as a negative control hole; drying the capillary to fix the primer on the inner wall of the capillary.

11. The method according to claim 9, wherein the step (2) is carried out by adding the sample to be detected, the LAMP reaction reagent and the color-developing agent into the reaction well of the microarray chip and reacting at 63 ℃ for 1 hour.

12. The method of claim 9, wherein the reaction result detection and data analysis in step (3) are specifically: after the LAMP reaction is finished, the color development condition of all reaction holes on the chip is detected through ultraviolet fluorescence, and the detected result is judged to be positive or negative under the condition that the positive control hole develops color and the negative control hole does not develop color.