Path:
Home >
MISC >
Page351 > WS 588-2018
Price & Delivery
US$449.00 · In stock · Download in 9 secondsWS 588-2018: Diagnosis for hand, foot and mouth disease
Delivery: 9 seconds. True-PDF full-copy in English & invoice will be downloaded + auto-delivered via email. See
step-by-step procedureStatus: Valid
| Std ID | Version | USD | Buy | Deliver [PDF] in | Title (Description) |
| WS 588-2018 | English | 449 |
Add to Cart
|
5 days [Need to translate]
|
Diagnosis for hand, foot and mouth disease
|
Click to Preview a similar PDF
Basic data
| Standard ID | WS 588-2018 (WS588-2018) |
| Description (Translated English) | Diagnosis for hand, foot and mouth disease |
| Sector / Industry | Health Industry Standard |
| Classification of Chinese Standard | C59 |
| Word Count Estimation | 19,151 |
| Date of Issue | 2018-03-06 |
| Date of Implementation | 2018-08-01 |
| Regulation (derived from) | State-Health-Communication (2018) 4 |
| Issuing agency(ies) | National Health Commission |
WS 588-2018: Diagnosis for hand, foot and mouth disease
---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Diagnosis for hand, foot and mouth disease
ICS 11.020
C 59
WS
People's Republic of China Health Industry Standard
Hand, foot and mouth disease diagnosis
Published on.2018-03-06
2018-08-01 implementation
National Health and Family Planning Commission of the People's Republic of China
Content
Foreword...II
1 Scope...1
2 Terms and definitions...1
3 Abbreviations...1
4 Diagnosis basis...1
5 Diagnostic principles... 2
6 Diagnosis...3
7 Differential diagnosis...3
Appendix A (Normative) Detection of enterovirus-specific antibodies related to hand, foot and mouth disease...5
Appendix B (Normative Appendix) Hand, Foot and Mouth Disease Related Intestinal Virus Nucleic Acid Detection...10
Appendix C (Normative Appendix) Isolation and Identification of Enteroviruses Related to Hand, Foot and Mouth Disease...14
Foreword
Chapter 6 of this standard is mandatory and the rest are recommended.
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard was drafted. Beijing Ditan Hospital affiliated to Capital Medical University, China Center for Disease Control and Prevention, and affiliated to Capital Medical University
Beijing Children's Hospital, China Center for Disease Control and Prevention, Institute of Viral Disease Prevention and Control.
The main drafters of this standard. Li Xingwang, Yang Weizhong, Wang Zijun, Qian Suyun, Chen Zhihai, Xu Wenbo, Zhang Jing, Lu Union.
Hand, foot and mouth disease diagnosis
1 Scope
This standard specifies the diagnosis basis, diagnosis principle, diagnosis and differential diagnosis of hand, foot and mouth disease.
This standard is applicable to the diagnosis of foot and mouth disease of various medical and health institutions at all levels and their medical staff.
2 Terms and definitions
The following terms and definitions apply to this document.
2.1
Hand, foot and mouth disease
Acute infectious diseases caused by enteroviruses such as human enterovirus 71 (EV-A71) and coxsackievirus group A (CV-A16) are more common.
For preschool children. Severe cases are mostly caused by EV-A71 infection.
3 Abbreviations
The following abbreviations apply to this document.
CPE. cytopathic effect
CV-A16. Coxsackievirus A group 16 (coxsackievirus A16)
cDNA. Complementary DNA
dNTP. deoxy-ribonucleotide triphosphate
EDTA. ethylenediaminetetraacetic acid
ELISA. enzyme-linked immunosorbent assay
EV-A71. human enterovirus type 71 (human enterovirus A71)
HEPES. 4-Hydroxyethylpiperazineethanesulfonic acid buffer
Hep-2. human laryngeal carcinoma epithelial cells
IgG. immunoglobulin G (immunoglobulin G)
IgM. immunoglobulin M (immunoglobulin M)
MEM. Eagle's minimum essential medium
OPD. o-phenylenediamine
RD. human rhabdomyosarcoma cells
RT-PCR. reverse transcription-polymerase chain reaction
TMB. 3,3',5,5'-tetramethylbenzidine
Vero. African Green Monkey Kidney Cells
4 diagnosis basis
4.1 Epidemiological characteristics
It can be ill all year round and has seasonal distribution characteristics. The main peaks in spring and summer and the peaks in autumn and winter can occur in the south.
It is popular in summer and autumn, especially in summer. Enterovirus is highly contagious, has a large proportion of latent infections, has a complicated transmission route, and has a fast spread speed.
In the meantime, a wide range of epidemics can be caused. During the epidemic period, clustering or outbreaks can occur in kindergartens, kindergartens, and families.
4.2 Clinical manifestations
The incubation period is generally 2 d to 10 d, with an average of 3 d to 5 d.
Acute onset, fever, maculopapular rash, herpes on the hands, feet and buttocks, scattered herpes in the oral mucosa or pharyngeal isthmus. May be accompanied by coughing,
Flowing, loss of appetite, diarrhea and other symptoms.
Some cases are only manifested as rashes of the hands, feet and buttocks and/or herpes of the pharynx. In a few cases, the rash is not typical, showing a small sand-like shape.
A rash, a single-site rash, or no rash.
A small number of cases can affect the central nervous system, manifested as meningitis, encephalitis, encephalomyelitis, and even pulmonary edema, pulmonary hemorrhage and/or
Circulatory dysfunction, etc., the disease progresses rapidly and can cause death.
4.3 Laboratory examination
4.3.1 General inspection
4.3.1.1 Blood routine
White blood cell counts are normal or decreased, and white blood cell counts can be elevated in severe and critical cases.
4.3.1.2 Blood sugar
Critical cases can be raised.
4.3.1.3 Cerebrospinal fluid
Central nervous system involvement often manifests as. clear appearance, increased pressure, increased white blood cell count, mostly mononuclear cells, protein
Normal or mild increase, sugar and chloride are normal.
4.3.2 Serology and pathogen examination
4.3.2.1 Detection of enterovirus IgM antibodies such as EV-A71 or CV-A16 in serum or cerebrospinal fluid by ELISA or the like (see
Appendix A).
4.3.2.2 ELISA, neutralization test and other methods to detect EV-A71 or CV-A16 and other enterovirus IgG antibodies in the serum, recovery period
Serum was ≥4 fold higher than the acute phase or negative for acute phase antibody and positive for antibody during recovery.
4.3.2.3 From the patient's nasopharyngeal swab, anal swab, feces, herpes fluid, cerebrospinal fluid or corpse by RT-PCR, fluorescence quantitative RT-PCR, etc.
Enterovirus-specific nucleic acids such as EV-A71 or CV-A16 were detected in the specimens (see Appendix B).
4.3.2.4 Using nasopharyngeal swabs, anal swabs, feces, herpes fluid, cerebrospinal fluid or autopsy specimens with cell lines such as RD, HEp-2 or Vero
For virus culture, enteroviruses such as EV-A71 or CV-A16 can be isolated (see Appendix C).
5 Diagnostic principles
5.1 Clinical diagnosis and classification can be made based on clinical manifestations and general laboratory findings. Epidemiological data can be used as a reference.
5.2 The diagnosis requires a positive result related to the etiology and serological tests of hand, foot and mouth disease.
6 diagnosis
6.1 Clinical diagnosis cases
Meets 4.2 and excludes other related diseases.
6.2 confirmed cases
Clinically diagnosed cases and have one of 4.3.2.
6.3 Clinical classification
6.3.1 Ordinary type
Hand, foot, buttocks rash, oral mucosal herpes, with or without fever.
6.3.2 Heavy duty
There are manifestations of central nervous system involvement, such as. mental disability, lethargy, convulsions, convulsions; headache, vomiting; limb shaking, myoclonus,
Ocular tremor, ataxia, ocular dyskinesia; weakness or acute flaccid paralysis; convulsions. Visible meningeal irritation, reduced sputum reflex or
disappear. Laboratory tests can have elevated white blood cells and blood sugar.
6.3.3 Critical
In severe cases, one of the following occurs.
a) frequent central nervous system damage such as convulsions, coma, and cerebral palsy;
b) respiratory dysfunction, bloody foam sputum, purpura and other respiratory dysfunction;
c) Circulatory dysfunction such as skin spots, cold limbs, markedly increased heart rate, and marked rise or fall in blood pressure.
7 differential diagnosis
7.1 Other rash diseases
The common type of hand, foot and mouth disease should be differentiated from diseases such as papular urticaria, chickenpox, atypical measles, childhood acute rash, herpes zoster and rubella.
Can be identified according to epidemiology, rash morphology, location, time and sequence of rash, lymph node enlargement and accompanying symptoms, etc.
The form and location of the rash is the most important. It can be identified based on the results of pathogens and serological tests.
7.2 Meningitis or encephalitis caused by other viruses
Meningitis or encephalitis caused by other viruses, such as herpes simplex virus, cytomegalovirus, Epstein-Barr virus, Japanese encephalitis virus, etc.
The clinical manifestations were similar to those of central nervous system lesions in hand, foot and mouth disease. Atypical to rash, according to epidemiological history, etiology or
The serological test results are diagnosed.
7.3 Polio
Acute flaccid paralysis (AFP) in hand, foot and mouth disease should be identified with polio. The latter is mainly characterized by bimodal fever, the second course of disease
Flaccid paralysis occurs before or during the antipyretic period, and there is no rash. Finally, it is identified based on the results of pathogenic tests.
7.4 Pneumonia
Pulmonary edema can occur in severe cases of hand, foot and mouth disease and should be differentiated from pneumonia. Pneumonia mainly manifests as fever, cough, shortness of breath, etc.
Symptoms, generally no rash; the disease progresses relatively slowly, the chest X-ray aggravation or alleviation gradually evolves, showing lung consolidation lesions, atelectasis and
Pleural effusion and so on.
Appendix A
(normative appendix)
Detection of enterovirus-specific antibodies related to hand, foot and mouth disease
A.1 ELISA for detection of EV-A71 IgM antibody
A.1.1 Test materials
The test materials are as follows.
a) microplate. coated with anti-human IgM;
b) antigen reagent. containing EV-A71 antigen;
c) enzyme labeling reagent. horseradish peroxidase-labeled EV-A71 antibody;
d) positive control. containing anti-EV-A71 IgM antibody positive serum;
e) Negative control. no anti-EV-A71 IgM antibody serum;
f) Specimen dilution. protein containing buffer;
g) concentrated washing liquid. containing not less than 2.5% of a surfactant;
h) substrate. TMB or OPD;
i) Stop solution. Contains sulfuric acid at a concentration of 2 mol/L.
A.1.2 Specimen
The specimens are as follows.
a) Serum or plasma, with or without specimens of anticoagulants such as EDTA, sodium citrate or heparin. No suspended fibrin or polymer,
Severe hemolysis and bacterial contamination.
b) Cerebrospinal fluid.
A.1.3 Steps
Due to the different methods and procedures of different kits, the specific operation is carried out according to the reagent instructions. This article takes the ELISA general operation as an example.
The detection steps are as follows.
a) The reagent is equilibrated at room temperature for more than 30 min;
b) dosing. dilute the concentrated washing solution with distilled water or deionized water 20 times;
c) No.. The microplates corresponding to the specimens are numbered sequentially, and each plate should have 3 holes for the negative control, 2 holes for the positive control and 1 hole for the blank control;
d) Add diluent. Add 100 μL of sample dilution to each well, except for blank wells;
e) Adding the sample to the corresponding well or the negative and positive control 10 μL, gently mix by shaking (cerebrospinal fluid can
1..2 dilution);
f) Incubation. after sealing with a sealing film, incubate at 37 ° C for 30 min;
g) Washing the board. Carefully remove the sealing film, wash it 5 times with a washing machine, and buckle it for the last time;
h) Add antigen. 50 μL of antigen reagent is added to each well, except for blank wells;
i) Add enzyme. add 50 μL of enzyme labeling reagent to each well, except for blank wells, gently shake and mix;
j) Incubation. After sealing with a sealing film, incubate at 37 ° C for 30 min;
k) Washing the board. carefully remove the sealing film, wash it 5 times with a washing machine, and buckle it for the last time;
l) Color development. Add 50 μL of each of the developer A and B solution to each well, gently shake and mix, and color at 37 ° C for 15 min;
m) Determination. Add 50 μL of stop solution to each well, mix gently by shaking, and measure the result within 10 min to set the wavelength of the microplate reader at 450 nm.
The OD value of each well was measured after zeroing with a blank hole.
A.1.4 Reagent control range
A.1.4.1 Negative control OD value ≤ 0.1. If the 1-hole negative control OD value > 0.1 should be discarded, if the 2-hole or 3-hole negative control OD value > 0.1, should be heavy
Repeat the test.
A.1.4.2 Positive control OD value ≥ 0.8.
A.1.5 Result judgment
A.1.5.1 Cutoff calculation. Threshold = 0.1 Negative control OD mean. (The negative control well OD value is less than 0.05 by 0.05
Count).
A.1.5.2 Negative judgment. The OD value of the specimen is < critical value, which is negative for EV-A71 IgM antibody.
A.1.5.3 Positive judgment. The OD value of the specimen is ≥ critical value and is positive for EV-A71 IgM antibody.
A.2 Detection of intestinal virus neutralizing antibodies in hand, foot and mouth disease
A.2.1 Liquid preparation
A.2.1.1 serum treatment solution (liquid A)
100 mL contains the following liquids.
a) MEM, 85 mL;
b) 3% L-glutamine, 1 mL;
c) 7.5% sodium bicarbonate, 2 mL;
d) fetal calf serum, 2 mL;
e) Cyan and streptomycin (10 000 U/mL each), 10 mL.
A.2.1.2 Cell nutrient solution (B solution)
Formulated as a growth solution with 100 mL of the following liquids.
a) MEM, 85 mL;
b) 3% L-glutamine, 1 mL;
c) 7.5% sodium bicarbonate, 2 mL;
d) HEPES, 1 mL;
e) fetal calf serum, 10 mL;
f) Penicillin, streptomycin (10 000 U/mL each), 1 mL.
A.2.1.3 Virus (serum) dilution (C solution)
Formulated as a maintenance solution, the following liquids are contained in 100 mL.
a) MEM, 93 mL;
b) 3% L-glutamine, 1 mL;
c) 7.5% sodium bicarbonate, 2 mL;
d) HEPES, 1 mL;
e) fetal calf serum, 2 mL;
f) Penicillin, streptomycin (10 000 U/mL each), 1 mL.
A.2.2 Attack virus CCID50 titration and titer gradient preparation (EV-A71 or CV-A16)
A.2.2.1 Freeze and thaw the proliferated virus suspension three times, then centrifuge at 10 °C for 12 min at 4 °C, and take the supernatant for dispensing.
In 20 frozen tubes, 0.5 mL per tube, kept in a -70 ° C refrigerator for long-term storage. Take one tube for each neutralization test, and each tube should be tested once.
When the test is used up, the rest should be inactivated and discarded.
A.2.2.2 Add 10 times of the Eagle solution to 10-8~10-1 virus solution, add into the cell plate, 50μL per well, 4 pores per dilution.
Cell.
A.2.2.3 Add 50 μL of cell suspension to each well, and set a cell control (50 μL of 50 μL cell suspension) and incubate at 36 ° C for 7 d.
Check for cell lesions.
A.2.2.4 Calculate the CCID50 of the isolated virus strain according to the Behrens-Kärber formula.
A.2.2.5 Before the formal test, the virus should be titrated 2 times to 3 times, and the average value should be taken to find the virus containing 100 CCID50 per 0.05 mL.
Load.
A.2.2.6 Prepare the challenge virus according to the calculated dilution ratio and determine the total amount of virus required for the test (ie 100 CCID50/0.05 mL).
A.2.2.7 Take 3 small tubes, each with a virus dilution (C solution in liquid preparation) 0.9 mL.
A.2.2.8 Use a filter tip with a filter tip (ART tip) to absorb 0.1 mL of diluted virus solution (ie 100 CCID50/0.05 mL) to
In the first small test tube, change the tip of the other ART and mix gently and thoroughly to avoid a large amount of aerosol. Dilute according to this method.
To 0.1 CCID50/0.05 mL.
A.2.3 Dilution serum
A.2.3.1 The acute phase serum collected from 1 day to 7 days after onset, the recovery period serum collected from 3 weeks to 4 weeks after onset, frozen at -20 °C
For inspection.
A.2.3.2 Take several small sterile test tubes (one for each serum) on a test tube rack, and add serum treatment solution to each tube (A.2.1.1 liquid with
In the system, the liquid A is 0.3 mL, and the serum to be tested is 0.1 mL. The stopper is tightly packed, shaken and mixed, and placed in a refrigerator at 4 ° C overnight, that is, the serum is diluted 1.4.
Inactivated at 56 ° C for 30 min the next day.
A.2.3.3 Open the independent sterile packaging 48-well tissue culture plate, use it vertically, and add serum dilution to each well (A.2.1.3 C in liquid preparation)
Liquid) 0.3 mL, one row per serum, 4 wells per row. Use a pipette to take 0.1 mL of the treated serum into the first well (ie 1.16).
Blow 8 times to 10 times, add 0.1 mL to the second hole (ie 1.64), and then to 1.1 024. It is not necessary to change the tip during the serum dilution.
That is, each serum sample was diluted 4 times, ie 1.4, 1.16, 1.64, 1.256, 1. 024.
A.2.3.4 Two dilutions should be made in parallel for each dilution of each serum sample.
A.2.4 Procedures for the determination of virus neutralizing antibodies
The specific steps are as follows.
a) Take a 96-well plate for lateral use. Each plate can be used for 8 (4 pairs) of serum to be tested. The layout is shown in Figure A.1.
1) Add 1.1 024 to each hole in the A1-A2 hole (B1-B2, C1-C2, D1-D2, E1-E2, F1-F2, G1-G2, H1-H2)
The dilution of the test serum is 0.05 mL, and it is not necessary to change the suction tip;
2) Add 1.256 to each well in A3-A4 wells (B3-B4, C3-C4, D3-D4, E3-E4, F3-F4, G3-G4, H3-H4)
Dilution of the test serum 0.05 mL;
3) Add 1.64 dilution to each well of A5-A6 well (B5-B6, C5-C6, D5-D6, E5-E6, F5-F6, G5-G6, H5-H6)
Degree of test serum 0.05 mL;
4) A7-A8 holes (B7-B8, C7-C8, D7-D8, E7-E8, F7-F8, G17-G8, H7-H8) with 1.16 dilution in each well
The serum to be tested is 0.05 mL;
5) Add each hole in A9-A10 well (B9-B10, C9-C10, D9-D10, E9-E10, F9-F10, G9-G10, H9-H10)
1.4 dilution of the test serum 0.05 mL;
6) A11-A12 holes (B11-B12, C11-C12, D11-D12, E11-E12, F11-F12, G11-G12, H11-H12) are
For each serum control well to be tested, add 0.05 mL of diluent to each well.
Figure A.1 Virus Neutralizing Antibody Assay 96-well Plate Layout
b) Add 0.05 mL of virus to the above wells (the virus titer has been diluted to 100 CCID50/0.05 mL).
c) After capping, mix with a microplate mixer and incubate for 2 h in a 36 ° C CO2 incubator.
d) Take another 96-well plate for longitudinal use and verify the virus titer of 100 CCID50/0.05 mL (must be done for each experiment).
Add 0.05 ml of virus dilution (C solution in reagent preparation) to each well, then add up from 0.1 CCID50/0.05 mL.
0.05 mL, 8 wells per dilution, no need to change the suction tip, always add to 100 CCID50/0.05 mL; while leaving 4 holes to do
For the cell control wells, add 0.1 mL of virus dilution to each well and store in a 4 °C freezer.
e) During the incubation period, digest the cells with digestive juice to prepare a cell suspension with a cell suspension concentration of 2 × 105 cells/mL, 96 wells per well.
The plate needs to be prepared at least 10 mL.
f) Each serum hole to be tested, serum control well (to be tested), virus retrophage and cell control well (virus back)
Add 0.1 mL of cell suspension to the drip plate, then mix with a microplate mixer and incubate in a 36 ° C CO2 incubator;
g) observe the CPE daily using an inverted microscope and record the virus titration results to the highest dilution of serum without cytopathic effects
The countdown is the end point titer. When 100 CCID50/0.05 mL of virus control wells showed complete lesions, the final result was determined (5 d~
7 d).
h) Note. If the virus control result (virus back drop) is not in the range of 32 CCID50/0.05 mL to 320 CCID50/0.05 mL,
If the experiment is invalid, repeat the experiment.
A.2.5 Determination of results
A.2.5.1 When one of the 2 wells of the highest dilution serum shows cytopathic effect and the other hole does not show cytopathic effect, the reciprocal of the dilution
That is, the neutralizing antibody titer of the serum sample.
A.2.5.2 When the high-dilution 2 wells are completely lesioned and the adjacent low-dilution 2 wells are completely free of lesions, the reciprocal of the average dilution of the two is the blood.
The neutralizing antibody titer of the clear specimen.
A.2.5.3 When there is 1 hole cytopathic effect in two adjacent dilution sera and no cytopathic effect in the other hole, the average dilution of the two is down.
The number is the neutralizing antibody titer of the serum sample.
A.2.5.4 For the double serum neutralization test results of patients with HFMD, if the recovery period serum is higher than the acute phase serum EV-A71 or CV-A16
The neutralizing antibody titer can be diagnosed by a 4-fold or more-fold increase.
A.2.5.5 If the recovery period serum is 4 times or more than 4 times higher than other acute enterovirus neutralizing antibody titers in the acute phase, the intestinal tract can be confirmed.
Whether the virus is infected or not should be combined with clinical and epidemiological judgments.
Appendix B
(normative appendix)
Hand, foot and mouth disease related enterovirus nucleic acid detection
B.1 RT-PCR detection of enterovirus nucleic acid
B.1.1 Test materials
B.1.1.1 Clinical specimens for detection of enterovirus in hand, foot and mouth disease. nasopharyngeal swabs, anal swabs, feces, herpes fluid, cerebrospinal fluid or corpse
Check the specimen, or its virus isolation culture.
B.1.1.2 Amplification primers are designed as follows.
a) Universal primer sequence for human enterovirus nucleic acid detection (product length 116 bp). PE2 (upstream). 5'- TCC GGC CCC TGA
ATG CGG CTA ATC C -3'; PE1 (downstream). 5'- ACA CGG ACA CCC AAA GTA GTC GGT CC -3';
b) EV-A71 nucleic acid detection primer sequence (product length 226 bp). EV-A71-S (upstream). 5'- GCA GCC CAA AAG AAC
TTC AC -3'; EV-A71-A (downstream). 5'- ATT TCA GCA GCT TGG AGT GC -3';
c) CV-A16 nucleic acid detection primer sequence (product length 208 bp). CV-A16-S (upstream). 5'-ATT GGT GCT CCC ACT
ACA GC-3'; CV-A16-A (downstream); 5'-TCA GTG TTG GCA GCT GTA GG-3'.
B.1.1.3 Total RNA extraction reagent. RNA can be extracted using a commercial kit.
B.1.1.4 AMV reverse transcriptase, thermostable DNA polymerase and dNTP.
B.1.2 Operation steps. RT-PCR operation and reaction procedure of different kits are different. The specific operation is subject to the manual. This article uses two-step method RT.
-PCR is an example.
B.1.2.1 Total RNA extraction. Extract the total RNA from the cells according to the reagent instructions to prepare template RNA.
B.1.2.2 Design Control, A. Positive Control. Inactivated virus control. B. Normal cell control. C. reagent control. replace with deionized water
specimen.
B.1.2.3 Reverse transcription synthesis cDNA. Complementary to the target gene RNA sequence by reverse transcription synthesis according to the AMV reverse transcriptase manufacturer's instructions
cDNA
B.1.2.4 Configuration reaction system. Different kit configuration systems are different. This article takes the general general method as an example. See Table B.1 for specific configuration.
Table B.1 PCR reaction system configuration (50 μL system)
Reagent name volume
10×PCR Buffer 5 μL
50 mM MgCl2 1.5 μL
10 mM dNTP Mix 1 μL
Upstream primer (0.1 μg/μL) 1 μL
Downstream primer (0.1 μg/μL) 1 μL
Taq DNA Polymerase (5 U/μL) 0.5 μL
cDNA (reverse transcription product) 5 μL
RNase Free dH2O· 35 μL
B.1.2.5 PCR amplification. specific amplification of the target gene by PCR cycle, reaction conditions. pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s,
Annealing at 50 ° C for 30 s, extending at 72 ° C for 40 s, a total of 30 ~ 35 cycles; the last 72 ° C extension for 10 min.
B.1.2.6 PCR amplification products were detected by 2% agarose gel electrophoresis.
B.1.3 Result judgment
If the molecular weight of the electrophoresis band of the PCR amplification product is the same as the expected product fragment size, it is positive. Laboratory diagnosis of specimens
Determine according to Table B.2.
Table B.2 PCR product results judgment reference table
Laboratory diagnostic results of RT-PCR results of specimens to be examined
EV(-), EV-A71(-), CV-A16(-) Enterovirus negative
EV (+), EV-A71 (-), CV-A16 (-) other enteroviruses other than EV-A71, CV-A16
EV(+), EV-A71(+), CV-A16(-) EV-A71
EV(+), EV-A71(-), CV-A16(+) CV-A16
B.1.4 Meaning
A positive result indicates a hand-foot-and-mouth disease-related enterovirus infection.
B.2 Fluorescence quantitative RT-PCR for detection of enterovirus nucleic acid
B.2.1 Method
Single channel detection EV-A71, single channel detection CV-A16, single channel detection of enterovirus; or dual channel simultaneous detection of EV-A71 and
CV-A16; or simultaneous detection of EV-A71 and CV-A16 and enterovirus using three channels.
B.2.2 Test materials
B.2.2.1 Test specimens. see B.1.1.1.
B.2.2.2 Amplification primers and fluorescent probes are designed as follows.
a) CAV16 fluorescent primer probe. upstream primer. CAV16YGF. 5'-GGGAATTTCTTTAGCCGTGC-3'; downstream primer. CAV16YGR.
5'-CCCATCAARTCAATGTCCC-3'; probe (FAM fluorescein label). CAV16YGPB. 5'-(FAM) –
ACAATGCCCACCACGGGTACACA-(BHQ1)-3'.
b) EV71 fluorescent primer probe. upstream primer. EV71YGF. 5'-TGATTGAGACACGCTGTGTTCTTA-3''; downstream primer.
EV71YGR. 5'-...
...
