CN117305516A - Kit for rapidly detecting hand-foot-and-mouth disease combined viral encephalitis - Google Patents

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit for rapidly detecting hand-foot-and-mouth disease combined viral encephalitis. The invention provides a primer probe composition for detecting pathogens related to hand-foot-mouth disease combined viral encephalitis, which comprises primers and probes of coxsackievirus A16, enterovirus 71 and encephalitis B virus, and can effectively detect the coxsackievirus A16, enterovirus 71 and encephalitis B virus simultaneously and carry out qualitative analysis. The method has the advantages of simple detection method, high sensitivity, strong specificity, good anti-interference capability, good stability and the like. Realizes the simultaneous detection of Coxsackie virus A16, enterovirus 71 and Japanese encephalitis virus by one tube of reaction liquid, and has wide application prospect.

Description

Kit for rapidly detecting hand-foot-and-mouth disease combined viral encephalitis

Technical Field

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit for rapidly detecting hand-foot-and-mouth disease combined viral encephalitis.

Background

Hand-foot-and-mouth disease (Hand foot mouth disease, HFMD) is a common infectious disease caused by enteroviruses, and is clinically characterized by hand, foot and mouth herpes, so that the disease is commonly called hand-foot-and-mouth disease. There are 20 or more enteroviruses that cause hand-foot-and-mouth disease, of which Coxsackie virus type A16 (Coxsackie-virus 16, CA 16) and enterovirus type 71 (Enter-virus 71, EV71) are most common. The disease is mainly transmitted through the digestive tract, respiratory tract, close contact and other ways. Clinically, fever, maculopapules and herpes at the ends of the mouth and limbs are the main manifestations, and meningitis, encephalitis, encephalomyelitis, pulmonary edema, circulatory disturbance and the like can occur in heavy people. The cause of mortality is mainly brain stem encephalitis and neurogenic pulmonary edema. Epidemic diseases are often caused by the strong infectivity of viruses. The inspection of the hand-foot-and-mouth disease is very critical for diagnosing the hand-foot-and-mouth disease, not only can judge the etiology and the disease severity of the hand-foot-and-mouth disease, but also has important effect on the subsequent treatment of the hand-foot-and-mouth disease.

Viral encephalitis (Viral encephalitis, VE) is a central nervous system infectious disease caused by invasion of the brain parenchyma by viruses, and the incidence rate is about 5/10 ten thousand at present, and frequently occurs in children. Viral encephalitis can also induce the body to present myelitis and brain stem encephalitis, and common symptoms and signs include convulsion, focal nervous symptoms, fever, headache, vomiting, neck rigidity, disturbance of consciousness and the like. The infant suffering from the hand-foot-mouth disease and encephalitis can cause the worsening of the disease, not only directly invade neurons, but also cause vascular endothelial cell permeability by releasing various inflammatory factors, thereby further damaging the nervous system. Epidemic encephalitis B is a common central nervous system infectious disease, caused by encephalitis B virus (Japanese encephalitis virus, JEV) infection, and has high mortality and disability rate. JEV infects and breaks through the blood brain barrier, enters the central nervous system, directly targets neurons and causes neuronal damage. At present, the factors related to the neuronal damage caused by JEV are considered to comprise inflammatory reaction, apoptosis, endoplasmic reticulum stress and the like, and the specific mechanism is not completely known.

It can be seen that coxsackievirus a16, enterovirus 71 and encephalitis b viruses can all be involved in the development of encephalitis diseases in a specific manner. For hand-foot-mouth disease, diagnosis is not difficult according to clinical symptoms and physical signs, especially typical rash distribution characteristics of oral cavity and hand-foot parts during large-scale flow. However, when the medicine is dispersed, clinical manifestations are similar to those of herpetic stomatitis, rubella and herpes simplex stomatitis, so that the diagnosis of clinicians is difficult. Similarly, the clinical manifestation of Japanese encephalitis is similar to that of a traditional Chinese medicine, such as a toxic bacillary dysentery, suppurative meningitis and tuberculous meningitis, and misdiagnosis is caused to cause misdiagnosis, so that adverse effects are caused for patients. Therefore, early diagnosis of hand-foot-mouth disease and viral encephalitis and judgment of infectivity are key to prevention and treatment of disease.

For detecting hand-foot-mouth disease and epidemic encephalitis B pathogens, a detection method for detecting multiple targets by one-tube reaction liquid, improving detection speed, and having the advantages of simple method, high detection rate and the like is to be developed.

Disclosure of Invention

In order to solve the problems, the invention provides a primer probe composition for detecting pathogens related to combined viral encephalitis of hand-foot-and-mouth disease, which comprises primers and probes of Coxsackie virus A16, enterovirus 71, japanese encephalitis virus and internal standards, and can effectively detect Coxsackie virus A16, enterovirus 71 and Japanese encephalitis virus simultaneously and carry out qualitative analysis.

In one aspect, the invention provides a primer probe composition for detecting pathogens related to hand-foot-and-mouth disease combined viral encephalitis, which comprises primers and probes for detecting coxsackievirus A16, enterovirus 71 and encephalitis B viruses;

the nucleotide sequences of the forward primer, the reverse primer and the probe of the coxsackievirus A16 are SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.2 in sequence;

the nucleotide sequences of the forward primer, the reverse primer and the probe of the enterovirus 71 are SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.5 in sequence;

the nucleotide sequences of the forward primer, the reverse primer and the probe of the Japanese encephalitis virus are SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.8 in sequence.

Specifically, the composition further comprises an internal standard primer and a probe; the nucleotide sequences of the forward primer, the reverse primer and the probe of the internal standard are SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 in sequence.

Specifically, the components of each composition are present in a mixed manner.

Specifically, the probes are all connected with fluorescent groups at the 5 'end and quenching groups at the 3' end, and the fluorescent groups on the probes are not interfered with each other.

Further specifically, the aforementioned 5' -end-labeled fluorophore of the probe may be selected from FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3 or CY5; the 3' -end-labeled quenching group may be selected from DABCYL, BHQ-1, BHQ-2, BHQ-3 or ECLIPE.

Preferably, the CA 16-labeled fluorophore is FAM; the EV 71-labeled fluorophore is HEX (or VIC); the JEV-labeled fluorophore is ROX; the fluorescent group of the internal standard label is CY5.

In another aspect, the invention provides a kit for detecting a plurality of pathogens, the kit comprising the primer probe composition described above.

Specifically, the kit can also comprise a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer solution, mg ²⁺ At least one of dNTPs, taq enzyme, RT enzyme or DEPC water.

Specifically, the kit further includes, but is not limited to: negative quality control and positive quality control.

Specifically, the pathogen is used for detecting Coxsackie virus A16, enterovirus 71 and/or Japanese encephalitis virus.

Specifically, the forward primer of the coxsackievirus A16 can be used at a concentration of 0.15 pmol/mu L to 0.50 pmol/mu L, the reverse primer can be used at a concentration of 0.15 pmol/mu L to 0.50 pmol/mu L, and the probe can be used at a concentration of 0.12 pmol/mu L to 0.30 pmol/mu L.

Preferably, the forward primer of the coxsackievirus A16 is used at a concentration of 0.25 pmol/. Mu.L, the reverse primer is used at a concentration of 0.25 pmol/. Mu.L, and the probe is used at a concentration of 0.125 pmol/. Mu.L.

Specifically, the forward primer of enterovirus 71 type can be used at a concentration of 0.15 pmol/mu L-0.50 pmol/mu L, the reverse primer can be used at a concentration of 0.15 pmol/mu L-0.50 pmol/mu L, and the probe can be used at a concentration of 0.12 pmol/mu L-0.30 pmol/mu L.

Preferably, the enterovirus type 71 forward primer is used at a concentration of 0.25 pmol/. Mu.L, the reverse primer is used at a concentration of 0.25 pmol/. Mu.L, and the probe is used at a concentration of 0.125 pmol/. Mu.L.

Specifically, the forward primer concentration of the Japanese encephalitis virus can be 0.15 pmol/mu L-0.50 pmol/mu L, the reverse primer concentration can be 0.15 pmol/mu L-0.50 pmol/mu L, and the probe concentration can be 0.12 pmol/mu L-0.30 pmol/mu L.

Preferably, the encephalitis B virus forward primer is used at a concentration of 0.25 pmol/mu L, the encephalitis B virus reverse primer is used at a concentration of 0.25 pmol/mu L, and the encephalitis B virus probe is used at a concentration of 0.125 pmol/mu L.

Specifically, the forward primer may be used at a concentration of 0.15 pmol/. Mu.L to 0.50 pmol/. Mu.L, the reverse primer may be used at a concentration of 0.15 pmol/. Mu.L to 0.50 pmol/. Mu.L, and the probe may be used at a concentration of 0.12 pmol/. Mu.L to 0.30 pmol/. Mu.L.

Preferably, the internal standard forward primer is used at a concentration of 0.25 pmol/. Mu.L, the reverse primer is used at a concentration of 0.25 pmol/. Mu.L, and the probe is used at a concentration of 0.125 pmol/. Mu.L.

Further specifically, the aforementioned 5' -end-labeled fluorophore of the probe may be selected from FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3 or CY5; the 3' -end-labeled quenching group may be selected from DABCYL, BHQ-1, BHQ-2, BHQ-3 or ECLIPE.

Preferably, the CA 16-labeled fluorophore is FAM; the EV 71-labeled fluorophore is HEX (or VIC); the JEV-labeled fluorophore is ROX; the fluorescent group of the internal standard label is CY5.

Specifically, the detection method comprises the following steps:

(1) Extracting or releasing nucleic acid of a sample to be tested;

(2) Carrying out RT-qPCR amplification on the nucleic acid in the step (1) by using the primer probe composition or the kit;

(3) And judging negative and positive according to the amplification curve threshold.

Specifically, the determination method in the step (3) is as follows:

(1) An S-shaped amplification curve exists, the Ct value is less than or equal to 40, and the positive result is judged;

(2) And (3) no S-type amplification curve or Ct value is more than 40, and the CY5 internal standard channel is positive and is judged to be negative.

The invention has the technical effects that:

(1) The detection method is simple, and can detect Coxsackie virus A16, enterovirus 71 and Japanese encephalitis virus simultaneously;

(2) The sensitivity is high, and 100% detection limits of coxsackievirus A16, enterovirus 71 and Japanese encephalitis virus are 400.0copies/mL;

(3) The specificity is strong, and the common intestinal bacteria are not subjected to cross reaction;

(4) Has good anti-interference performance and stability.

Drawings

FIG. 1 is a graph showing the results of pathogen detection in example 1.

FIG. 2 is a graph showing the results of the type A16 sensitivity test for Coxsackie virus according to example 4.

FIG. 3 is a graph showing the results of enterovirus type 71 sensitivity test in example 4.

FIG. 4 is a graph showing the sensitivity test results of Japanese encephalitis virus in example 4.

FIG. 5 is a graph showing the detection results of the sensitivity of the internal standard in example 4.

FIG. 6 is a graph showing the results of a specific test of the composition of the present invention.

FIG. 7 is a graph showing the results of the detection of the composition of comparative example 1.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

The primer probe compositions used in the present invention are shown in Table 1.

TABLE 1

Wherein, the fluorescence reporter group of the CA16 probe is FAM; the fluorescence reporter group of the EV71 probe is HEX (or VIC); the fluorescent reporter group of JEV is ROX; the fluorescent reporter group of the internal standard is CY5.

Example 1 pathogen detection

1.1 reagents

The fluorescent PCR amplification reaction solution contains PCR buffer, mg2+, dNTP (T) s, taq enzyme, 0.1% DEPC water, primers, probes and the like. The reaction system in this example is shown in Table 2:

TABLE 2

Preparing an enzyme mixed solution: the enzyme mixture was composed of 1. Mu. L Anstart Taq DNAPolymerase enzyme (15U/. Mu.L) and 1. Mu. L Neoscript RTase enzyme (20U/. Mu.L) (2. Mu.L/human); neoscript RTase (20U/. Mu.L) was obtained by diluting Neoscript RTase (200U/. Mu.L) 10-fold; anstart Taq DNA Polymerase (15U/. Mu.L) was purchased from Guangdong Pengpeng Biotechnology Co., ltd. Neoscript RTase (200U/. Mu.L) was purchased from Zhuhai Baozhen Biotechnology Co., ltd.).

1.2 detection method

1.2.1 sample handling

Sample to be measured: cerebrospinal fluid of encephalitis patients in the morbidity stage is collected and directly placed in a preservation tube for preservation.

Negative control: the main component is normal saline.

Positive control: plasmids of the relevant primer probe region (Biotechnology (Shanghai) Inc.) were synthesized.

(1) 200. Mu.L of the sample to be tested, negative control, positive control were placed in a 1.5mL centrifuge tube, and nucleic acid extraction was performed using the nucleic acid extraction or purification reagent from St.Job's Biotechnology Co., ltd.

(2) And taking corresponding amounts of the PCR reaction liquid and the enzyme mixed liquid according to the quantity of the sample to be detected, the positive control and the negative control according to a proportion (38 mu L of the PCR reaction liquid/2 mu L of the enzyme mixed liquid), fully and uniformly mixing the PCR reaction liquid and the enzyme mixed liquid to 40 mu L of the PCR mixed liquid, and centrifuging at 2000rpm for 10s for later use.

(3) And (3) sucking 10 mu L of each of the treated sample, the negative control and the positive control, respectively adding the 10 mu L of each of the treated sample, the negative control and the positive control into a corresponding 0.2mL PCR reaction tube, adding 40 mu L of PCR mixed solution into each tube, and covering a tube cover.

1.2.2PCR amplification

And (3) performing PCR amplification on a PCR instrument of the SLAN-96P full-automatic medical PCR analysis system according to a certain temperature and time setting program. The program settings are shown in Table 3.

TABLE 3 Table 3

1.2.3 results and analysis

Samples FAM, HEX (VIC), ROX and channels have obvious S-shaped amplification curves, and Ct value is less than or equal to 40, and the samples are judged positive; samples FAM, HEX (VIC), ROX, channel No amplification curve (No Ct) or Ct value > 40, and CY5 internal standard channel is positive (Ct value less than or equal to 40), and the sample is judged to be negative. See in particular table 4:

TABLE 4 Table 4

The detection results are shown in fig. 1, and the CT values of all channels are 31-33, and the interpretation rule shows that the primer probe combination can detect Coxsackie virus A16, enterovirus 71 and Japanese encephalitis virus simultaneously.

Example 2 sensitivity test

The sensitivity of samples of different concentrations was tested using the test method of example 1, and the sample concentrations and test results are shown in table 5.

TABLE 5

The result shows that the 100% detection limit of the kit on coxsackievirus A16, enterovirus 71 and Japanese encephalitis virus is 400.0copies/mL (see figures 2-5).

Example 3 specificity test

The test results of the test conducted on common intestinal bacteria (bacteroides fragilis, helicobacter pylori, salmonella, staphylococcus aureus, vibrio parahaemolyticus, shigella, adenovirus, listeria monocytogenes, etc.) by the test method of example 1 are shown in fig. 6. The results show that the primer probe composition of the invention has no cross reaction to the enterobacteria. The determination results are shown in Table 6:

TABLE 6

Approximating pathogens	Detection result
		Mumps virus	Negative of
Rotavirus	Negative of
		Salmonella bacteria	Negative of
Staphylococcus aureus	Negative of
		Vibrio parahaemolyticus	Negative of
Cytomegalovirus	Negative of
		Adenovirus	Negative of
Listeria monocytogenes	Negative of

Example 4 interference immunity and stability test

4.1 interference immunity

The effect of the interferons on the assay of example 1 was analyzed using conventional methods, and experimental results showed that the potential PCR inhibitors/interfering substances such as dexamethasone (50. Mu.g/mL), cefmenoxime hydrochloride (50. Mu.g/mL), zanamivir (100. Mu.g/mL), ribavirin (100. Mu.g/mL), azithromycin (100. Mu.g/mL), histamine hydrochloride (200. Mu.g/mL), beclomethasone (50. Mu.g/mL), mupirocin (50. Mu.g/mL), tobramycin (50. Mu.g/mL), mometasone (50. Mu.g/mL), fluticasone (50. Mu.g/mL), budesonide (50. Mu.g/mL), triamcinolone acetonide (100. Mu.g/mL), heme (10. Mu.g/mL), purified mucin (20. Mu.g/mL), absolute ethanol (20% V/V) had no significant effect on the assay. See in particular table 7:

TABLE 7

4.2 stability

All reagents used in the detection method of example 1 were prepared as a kit in the amounts and proportions.

The stability of the kit was tested according to the conventional method, and the results showed that: the kit is detected after being stored for 11 months under the actual storage condition (-20+/-5 ℃), and has stable performance; the test result of the accelerated stability of the damage at 37 ℃ shows that the kit is stored in a constant temperature box at 37 ℃ for 24 hours, and the result meets the quality requirement; the freeze thawing stability test shows that the different kit passes through freeze thawing once at each detection time point at the actual storage temperature, and is continuously detected for 4 times, and the results all meet the quality requirements.

Comparative example 1

The invention also designs other primers and probes (the sequences of which are shown in table 8) to form different detection systems which are also used for joint detection of Coxsackie virus A16, enterovirus 71 and Japanese encephalitis virus. The method of reference example 1 was used for coxsackievirus a16, enterovirus 71 and encephalitis b virus. The specific detection result is shown in fig. 7, the Ct value detected is trailing and the fluorescence increment is obviously reduced, and the overall effect of the detection is poor. The results show that the overall detection effect of other primer probe sets is poor.

TABLE 8

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Claims (10)

1. The primer probe composition for detecting the pathogen related to the combined viral encephalitis of the hand-foot-and-mouth disease is characterized by comprising primers and probes for detecting coxsackievirus A16, enterovirus 71 and encephalitis B virus;

the nucleotide sequences of the forward primer, the reverse primer and the probe for detecting the coxsackievirus A16 are SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.2 in sequence;

the nucleotide sequences of the forward primer, the reverse primer and the probe for detecting enterovirus 71 are SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.5 in sequence;

the nucleotide sequences of the forward primer, the reverse primer and the probe for detecting the Japanese encephalitis virus are SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.8 in sequence.

2. The primer probe composition of claim 1, further comprising an internal standard primer and probe; the nucleotide sequences of the forward primer, the reverse primer and the probe of the internal standard are SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 in sequence.

3. Primer probe composition according to claim 1 or 2, characterized in that the components of each composition are present in a mixed manner.

4. A kit for detecting a pathogen associated with a viral encephalitis complicated by hand-foot-and-mouth disease, comprising the primer probe composition of any of claims 1-2.

5. The kit of claim 4, further comprising a nucleic acid releasing reagent, a nucleic acid extracting reagent, a PCR buffer, mg ²⁺ At least one of dNTPs, taq enzyme, RT enzyme or DEPC water.

6. The kit of claim 4, further comprising a negative quality control and a positive quality control.

7. The kit of claim 4, wherein the pathogen is a coxsackievirus a16, enterovirus 71, and/or encephalitis b virus.

8. The kit according to any one of claims 4 to 7, wherein the forward primer of coxsackievirus a16 is used at a concentration of 0.15 pmol/-0.50 pmol/. Mu.l, the reverse primer is used at a concentration of 0.15 pmol/. Mu.l-0.50 pmol/. Mu.l, and the probe is used at a concentration of 0.12 pmol/. Mu.l-0.30 pmol/. Mu.l;

the enterovirus 71 type forward primer has the concentration of 0.15 pmol/mu L-0.50 pmol/mu L, the reverse primer has the concentration of 0.15 pmol/mu L-0.50 pmol/mu L, and the probe has the concentration of 0.12 pmol/mu L-0.30 pmol/mu L;

the concentration of the forward primer of the Japanese encephalitis virus is 0.15 pmol/mu L-0.50 pmol/mu L, the concentration of the reverse primer of the Japanese encephalitis virus is 0.15 pmol/mu L-0.50 pmol/mu L, and the concentration of the probe of the Japanese encephalitis virus is 0.12 pmol/mu L-0.30 pmol/mu L;

the concentration of the forward primer and the reverse primer of the internal standard are respectively 0.15-0.50 pmol/mu L and 0.12-0.30 pmol/mu L, respectively.

9. The kit of claim 8, wherein the 5' end-labeled fluorophore of the probe is selected from FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY, and CY5; the 3' -end labeled quenching group is selected from DABCYL, BHQ-1, BHQ-2, BHQ-3 or ECLIPE.

10. The kit of claim 4, wherein the detection method comprises:

(1) Extracting or releasing nucleic acid of a sample to be tested;

(2) Performing RT-qPCR amplification of the nucleic acid of step (1) using the primer probe composition of any one of claims 1-3 or the kit of any one of claims 4-9;

(3) And judging negative and positive according to the amplification curve threshold.