CN102653794A - Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae - Google Patents
Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae Download PDFInfo
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- CN102653794A CN102653794A CN2012101486130A CN201210148613A CN102653794A CN 102653794 A CN102653794 A CN 102653794A CN 2012101486130 A CN2012101486130 A CN 2012101486130A CN 201210148613 A CN201210148613 A CN 201210148613A CN 102653794 A CN102653794 A CN 102653794A
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Abstract
The invention discloses a multiple touchdown PCR (polymerase chain reaction) detection kit capable of quickly detecting haemophilus influenzae in lower respiratory tract specimens, belongs to the technical field of molecular diagnosis of infectious diseases of lower respiratory tracts and aims to solve quick diagnosis of haemophilus influenzae in lower respiratory tract specimens. The kit comprises 10*PCR buffer solution, MgCl2, dNTP, TaqDNA polymerase, BSA (bovine serum albumin), haemophilus influenzae positive control DNA and three pairs of haemophilus influenzae primers. The invention discloses the multiple touchdown PCR detection kit and a detection method capable of detecting haemophilus influenzae and adopts agarose gel electrophoresis to detect the PCR products. The kit and the method have the advantages of quickness, simpleness and convenience, specificity and sensitivity and can be used for quick diagnosis and epidemiological survey of haemophilus influenzae infection.
Description
Technical field
The present invention relates to the molecular diagnostic techniques field that hemophilus influenzae infects, be specifically related to the application in hemophilus influenzae (Haemophilus influenzae) detection in the lower respiratory tract sample of multiple touchdown PCR detection method.
Background technology
Hemophilus influenzae is the The main pathogenic fungi that causes community acquired pneumonia, is severe bacteria, and it is cultivated needs the V factor and the X factor.Mainly cause pneumonia and meningitis, cause every year about 3,000,000 people serious ill and cause about 38.6 ten thousand people dead.Most of patients is children below 5 years old.The death that most hemophilus influenzaes cause betides developing country, and these countries do not include the hemophilus influenzae vaccine in the conventional inside the plan immunity (China is at present also for outside the plan) as yet.
The Hazard Factor that the aggressive hemophilus influenza pneumonia takes place the adult have: the severe pneumonia that immunocompromised, COPD, mellitus, cancer, splenectomy and chronic alcoholism, streptococcus aureus or influenza A virus cause.
The generation of the affecting conditions that vaccine inoculation can prevent to be caused by b type hemophilus influenzae.Developed country is owing to the extensive inoculation of vaccine, and children's hemophilus influenza pneumonia is eliminated.But do not promoting the country of vaccine inoculation, still can cause hundreds thousand of children to die from hemophilus influenzae in the year and infect the disease that causes.
(Guma M.K.Abdeldaim such as Guma M.K.Abdeldaim; Et al.; Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.Diagn Microbiol Infect Dis; 2009; 64 (4): 366-373.) having set up with omp P6 gene is the real-time quantitative PCR that target detects hemophilus influenzae, yet they find that this method can be accredited as hemophilus influenzae with other bacterial classification mistake of part influenzae.A plurality of genes to hemophilus influenzae detect the specificity that can improve detection method simultaneously.Korbie; (Korbie such as D.J.; D.J.and J.S.Mattick; Touchdown PCR for increased specificity and sensitivity in PCR amplification.Nat.Protocols, 2008.3 (9): p.1452-1456.) find specificity and the sensitivity that touchdown PCR can improve PCR.Current still do not have simultaneously to hemophilus influenzae omp P6, frdB, 16S rRNA gene to detect the multiplex PCR report of hemophilus influenzae.The multiple touchdown PCR that the present invention sets up can directly detect the hemophilus influenzae in the lower respiratory tract sample, and is time-consuming few, and tool high degree of specificity and susceptibility help quick diagnosis and epidemiology survey that hemophilus influenzae infects.
Summary of the invention
The technical problem that the present invention will solve provide a kind of can quick special detection lower respiratory tract sample in multiple PCR detection kit and the detection method of hemophilus influenzae.
In order to solve the problems of the technologies described above, the present invention realizes through following technological incidence of criminal offenses:
The multiple touchdown PCR detection kit of a kind of hemophilus influenzae comprises: 10 * PCR damping fluid, MgCl2, dNTP, TaqDNA polysaccharase, BSA, hemophilus influenzae positive control dna, 3 pairs of hemophilus influenzae primers;
The 1st pair: 5 '-GAAATGGTGCTGCTCAAACTTT-3 ' (SEQ ID NO.1) and 5 '-TGCGATGTTGTATTCTGGTGTA-3 ' (SEQ ID NO.2);
The 2nd pair: 5 '-CGATCAAGAGCCACATTTAAGC-3 ' (SEQ ID NO.3) and 5 '-CTTCTGAGCAATAGCCAACGA-3 ' (SEQ ID NO.4);
The 3rd pair: 5 '-CGTATTATCGGAAGATGAAAGTGC-3 ' (SEQ ID NO.5) (Hendolin PH; Et al.Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.J Clin Microbiol; 1997,35 (11): 2854-2858.) with 5 '-AGGATGTCAAGAGTAGGTAAGGTT-3 ' (SEQ IDNO.6);
The invention also discloses the detection method of using the mentioned reagent box:
(1) extracts sample DNA to be checked
(2) omp P6, frdB, the 16S rRNA gene in the multiple touchdown PCR method augmentation detection sample DNA to be checked, concrete steps are following:
3 pairs of hemophilus influenzae primers are:
Hi_omp?P6-F：5’-GAAATGGTGCTGCTCAAACTTT-3’（SEQ?ID?NO.1）
Hi_omp?P6-R：5’-TGCGATGTTGTATTCTGGTGTA-3’（SEQ?ID?NO.2）
Hi_frdB-F：5’-CGATCAAGAGCCACATTTAAGC-3’（SEQ?ID?NO.3）
Hi_frdB-R：5’-CTTCTGAGCAATAGCCAACGA-3’（SEQ?ID?NO.4）
Hi_16S?rRNA-F：5’-CGTATTATCGGAAGATGAAAGTGC-3’（SEQ?ID?NO.5）
Hi_16S?rRNA-R：5’-AGGATGTCAAGAGTAGGTAAGGTT-3’（SEQ?ID?NO.6）
(3) PCR reaction cycle parameter is following:
94 ℃ of 5min; 94 ℃ of 30s, 65 ℃ of (every circulation reduces by 0.5 ℃) 30s, 72 ℃ of 1min,
20 circulations; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 20 circulations; 72 ℃ of 7min.
(4) electrophoresis detection is identified:
Multiple touchdown PCR reaction product is carried out agarose electrophoresis detects, as contain in the electrophorogram in 215bp, 620bp, the 821bp band any 2 or 3 then representative detect hemophilus influenzae.
Beneficial effect: the multiple touchdown PCR that the present invention set up can overcome problems such as conventional length consuming time, sensitivity of cultivating is low, has characteristics such as quick, easy, special, sensitivity height.Can go out test results report the same day, can detect 3 genes of hemophilus influenzae simultaneously, the detection lower limit of hemophilus influenzae DNA was reached 0.2pg.This multiple touchdown PCR can be used for quick diagnosis and the epidemiology survey that hemophilus influenzae infects.
Description of drawings
Fig. 1 is the specific detection result of the multiple touchdown PCR of foundation; Swimming lane 1-12 template corresponds to successively: ETEC (Escherichia coli); Streptococcus aureus (Staphylococcus aureus); Pseudomonas aeruginosa (Pseudomonas aeruginosa); Klebsiella Pneumoniae (Klebsiella pneumoniae); Acinetobacter bauamnnii (Acinetobacter baumannii); Block its Moraxella (Moraxella (Branhamella) catarrhalis); Enterococcus faecalis (Enterococcus faecalis); M. smegmatics (Mycobacterium smegmatis); Streptococcus pneumoniae (Streptococcus pneumoniae); Mycobacterium tuberculosis (Mycobacterium tuberculosis); Mycobacterium bovis (Mycobacterium bovis); Mycobacterium bovis BCG (Mycobacterium bovisBCG); Swimming lane 13 is: DL2000DNA molecular mass standard; Swimming lane 14-16 template is followed successively by: positive control hemophilus influenzae (Haemophilus influenzae), b type hemophilus influenzae, negative control.
Fig. 2 is embodiment 1 detected result; Swimming lane M is a DL2000DNA molecular mass standard; Swimming lane 1-10 template is clinical sputum specimen genomic dna; The band of 2 corresponding sizes appears in swimming lane 5 in 215bp, 821bp place; The band of 3 corresponding sizes appears in swimming lane 6 in 215bp, 620bp, 821bp place; Be judged to and detect hemophilus influenzae, the bacteria cultivation results of its corresponding sample also confirms to detect hemophilus influenzae; Swimming lane 11,12 positive contrast and negative controls.
Fig. 3 is the detectability detected result of the multiple touchdown PCR of foundation: swimming lane 1-7 template corresponds to b type hemophilus influenzae genomic dna 20ng and 10 successively
-1 ~-6The dilution template; Swimming lane 8 is a DL2000DNA molecular mass standard; Swimming lane 9 negative contrasts; Detect down and be limited to 5 road templates, correspond to 2pgb type hemophilus influenzae genomic dna.
Embodiment
Embodiment 1:
The sputum specimen pre-treatment: sputum specimen is used saline water washing 2 times, and 1% pancreatin (at present joining existing usefulness the same day) that adds equivalent is in 37 ℃ of digestion 90min.
Extract the DNA of bacteria in the sputum specimen: get the sputum specimen 1.5ml that has digested and extract the DNA of bacteria in test kit (Takara, Dalian is precious gives birth to) the extraction sputum specimen with Takara minibest bacterial genomes.The dna direct that extracts is used for pcr amplification or places-20 ℃ of preservations subsequent use.
The total system 25 μ l of pcr amplification add 12.55 μ l distilled waters, 2.5 μ l damping fluids (10 * PCR) successively in 200 μ l PCR tubules; BSA (10mg/ml) 0.25 μ l, primer Hi_omp P6-F (10 μ mol/L) 0.5 μ l, Hi_omp P6-R (10 μ mol/L) 0.5 μ l, Hi_frdB-F (10 μ mol/L) 1 μ l; Hi_frdB-R (10 μ mol/L) 1 μ l; Hi_16S rRNA-F (10 μ mo/L) 1 μ l, Hi_16S rRNA-R (10 μ mol/L) 1 μ l, 2 μ lMgCl
2(25mM), 0.5 μ l dNTP (10mM), 0.2 μ l Taq archaeal dna polymerase (5U/ μ l), the sputum specimen genomic dna that 2 μ l extract.Establish hemophilus influenzae positive template contrast and negative control (distilled water is as template) simultaneously.Finger flicks mixing, of short durationly puts into the PCR appearance after centrifugal.
3 pairs of hemophilus influenzae primers are:
To Hi_omp the P6-F:5 '-GAAATGGTGCTGCTCAAACTTT-3 ' of hemophilus influenzae omp P6 gene design, Hi_omp P6-R:5 '-TGCGATGTTGTATTCTGGTGTA-3 ', amplification fragment length 215bp;
To the Hi_frdB-F:5 '-CGATCAAGAGCCACATTTAAGC-3 ' of hemophilus influenzae frdB gene design, Hi_frdB-R:5 '-CTTCTGAGCAATAGCCAACGA-3 ', amplification fragment length 620bp;
To Hi_16S the rRNA-F:5 '-CGTATTATCGGAAGATGAAAGTGC-3 ' of hemophilus influenzae 16S rRNA gene design, Hi_16S rRNA-R: 5 '-AGGATGTCAAGAGTAGGTAAGGTT-3 ', amplification fragment length 821bp.
PCR reaction cycle parameter is set: 94 ℃ of 5min; 94 ℃ of 30s, 65 ℃ of (every circulation reduces by 0.5 ℃) 30s, 72 ℃ of 1min, 20 circulations; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 20 circulations; 72 ℃ of 7min.
Amplified production carries out 2% agarose gel electrophoresis; Applied sample amount is to add behind 5 μ l and the 6 * sample-loading buffer mixing in the glue hole; In 1 * TAE solution, under 100V voltage, electrophoresis 30min; Electrophoresis is after behind ethidium bromide soaking and dyeing 10 ~ 20min of 0.5mg/L, analyzing and testing result in the gel imaging analysis system.Do not have band like negative control, positive control the band of corresponding size occurs in 215bp, 620bp, 821bp place, detect sample occur in 215bp, 620bp, the 821bp band any 2 or 3 then representative detect the hemophilus influenzae (see figure 2); Non-specific band appears in the band or the negative control that do not occur corresponding size like positive control in 215bp, 620bp, 821bp place, then is judged to the failure of an experiment, needs to detect again.
Embodiment 2:
The specific detection of multiple touchdown PCR.
Extract ETEC, streptococcus aureus, Pseudomonas aeruginosa, Klebsiella Pneumoniae, Acinetobacter bauamnnii, block the genomic dna of its Moraxella, enterococcus faecalis, M. smegmatics, streptococcus pneumoniae, mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, hemophilus influenzae and b type hemophilus influenzae; The pure growth of getting above-mentioned bacterium extracts test kit (Takara, Dalian is precious gives birth to) with the Takaraminibest bacterial genomes and extracts its genomic dna.The dna direct that extracts is used for pcr amplification or places-20 ℃ of preservations subsequent use.
The total system 25 μ l of pcr amplification add 12.55 μ l distilled waters, 2.5 μ l damping fluids (10 * PCR) successively in 200 μ l PCR tubules; BSA (10mg/ml) 0.25 μ l, primer Hi_omp P6-F (10 μ mol/L) 0.5 μ l, Hi_omp P6-R (10 μ mol/L) 0.5 μ l, Hi_frdB-F (10 μ mol/L) 1 μ l; Hi_frdB-R (10 μ mol/L) 1 μ l; Hi_16S rRNA-F (10 μ mol/L) 1 μ l, Hi_16S rRNA-R (10 μ mol/L) 1 μ l, 2 μ lMgCl
2(25mM), 0.5 μ l dNTP (10mM), 0.2 μ l Taq archaeal dna polymerase (5U/ μ l), template is the bacterial genomes DNA that 2 μ l extract.Establish negative control (distilled water is as template) simultaneously.Finger flicks mixing, of short durationly puts into the PCR appearance after centrifugal.
3 pairs of hemophilus influenzae primers are:
To Hi omp the P6-F:5 '-GAAATGGTGCTGCTCAAACTTT-3 ' of hemophilus influenzae omp P6 gene design, Hi_omp P6-R:5 '-TGCGATGTTGTATTCTGGTGTA-3 ', amplification fragment length 215bp;
To the Hi_frdB-F:5 '-CGATCAAGAGCCACATTTAAGC-3 ' of hemophilus influenzae frdB gene design, Hi_frdB-R:5 '-CTTCTGAGCAATAGCCAACGA-3 ', amplification fragment length 620bp;
To Hi_16S the rRNA-F:5 '-CGTATTATCGGAAGATGAAAGTGC-3 ' of hemophilus influenzae 16S rRNA gene design, Hi_16S rRNA-R: 5 '-AGGATGTCAAGAGTAGGTAAGGTT-3 ', amplification fragment length 821bp.
PCR reaction cycle parameter is set: 94 ℃ of 5min; 94 ℃ of 30s, 65 ℃ of (every circulation reduces by 0.5 ℃) 30s, 72 ℃ of 1min, 20 circulations; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 20 circulations; 72 ℃ of 7min.
Amplified production carries out 2% agarose gel electrophoresis; Applied sample amount is to add behind 5 μ l and the 6 * sample-loading buffer mixing in the glue hole; In 1 * TAE solution, under 100V voltage, electrophoresis 30min; Electrophoresis is after behind ethidium bromide soaking and dyeing 10 ~ 20min of 0.5mg/L, analyzing and testing result in the gel imaging analysis system.Hemophilus influenzae occurs corresponding big or small band with b type hemophilus influenzae in 215bp, 620bp, 821bp place, and other bacterial genomes template and negative control do not have band and (see figure 1) occurs.
Embodiment 3:
The detectability of multiple touchdown PCR is confirmed.
Extract the genomic dna of b type hemophilus influenzae: get b type hemophilus influenzae pure culture liquid 1.5ml and extract its genomic dna with Takaraminibest bacterial genomes extraction test kit (Takara, Dalian is precious gives birth to).The dna direct that extracts is used for pcr amplification or places-20 ℃ of preservations subsequent use.
The total system 25 μ l of pcr amplification add 12.55 μ l distilled waters, 2.5 μ l damping fluids (10 * PCR) successively in 200 μ l PCR tubules; BSA (10mg/ml) 0.25 μ l, primer Hi_omp P6-F (10 μ mol/L) 0.5 μ l, Hi_omp P6-R (10 μ mol/L) 0.5 μ l, Hi_frdB-F (10 μ mol/L) 1 μ l; Hi_frdB-R (10 μ mol/L) 1 μ l; Hi_16S rRNA-F (10 μ mol/L) 1 μ l, Hi_16S rRNA-R (10 μ mol/L) 1 μ l, 2 μ lMgCl
2(25mM), 0.5 μ l dNTP (10mM), 0.2 μ l Taq archaeal dna polymerase (5U/ μ l), template is the b type hemophilus influenzae genomic dna that extracts of 2 μ l or its 10
-1 ~-6The dilution template.Establish negative control (distilled water is as template) simultaneously.Finger flicks mixing, of short durationly puts into the PCR appearance after centrifugal.
3 pairs of hemophilus influenzae primers are:
To Hi omp the P6-F:5 '-GAAATGGTGCTGCTCAAACTTT-3 ' of hemophilus influenzae omp P6 gene design, Hi_omp P6-R:5 '-TGCGATGTTGTATTCTGGTGTA-3 ', amplification fragment length 215bp;
To the Hi_frdB-F:5 '-CGATCAAGAGCCACATTTAAGC-3 ' of hemophilus influenzae frdB gene design, Hi_frdB-R:5 '-CTTCTGAGCAATAGCCAACGA-3 ', amplification fragment length 620bp;
To Hi_16S the rRNA-F:5 '-CGTATTATCGGAAGATGAAAGTGC-3 ' of hemophilus influenzae 16S rRNA gene design, Hi_16S rRNA-R: 5 '-AGGATGTCAAGAGTAGGTAAGGTT-3 ', amplification fragment length 821bp.
PCR reaction cycle parameter is set: 94 ℃ of 5min; 94 ℃ of 30s, 65 ℃ of (every circulation reduces by 0.5 ℃) 30s, 72 ℃ of 1min, 20 circulations; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 20 circulations; 72 ℃ of 7min.Amplified production carries out 2% agarose gel electrophoresis; Applied sample amount is to add behind 5 μ l and the 6 * sample-loading buffer mixing in the glue hole; In 1 * TAE solution, under 100V voltage, electrophoresis 30min; Electrophoresis is after behind ethidium bromide soaking and dyeing 10 ~ 20min of 0.5mg/L, analyzing and testing result in the gel imaging analysis system.The minimum b type hemophilus influenzae genomic dna concentration that will occur corresponding size strip in 215bp, 620bp, 821bp place is defined as the detection lower limit (see figure 3) of this multiple touchdown PCR.
SEQUENCE?LISTING
< 110>Jiangsu University
< 120>the multiple touchdown PCR detection kit of hemophilus influenzae
<130>
<160> 6
<170> PatentIn?version?3.3
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<211> 22
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< 213>artificial sequence
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gaaatggtgc?tgctcaaact?tt 22
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<212> DNA
< 213>artificial sequence
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tgcgatgttg?tattctggtg?ta 22
<210> 3
<211> 22
<212> DNA
< 213>artificial sequence
<400> 3
cgatcaagag?ccacatttaa?gc 22
<210> 4
<211> 21
<212> DNA
< 213>artificial sequence
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cttctgagca?atagccaacg?a 21
<210> 5
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<212> DNA
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cgtattatcg?gaagatgaaa?gtgc 24
<210> 6
<211> 24
<212> DNA
< 213>artificial sequence
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aggatgtcaa?gagtaggtaa?ggtt 24
Claims (1)
1. the multiple touchdown PCR detection kit of hemophilus influenzae comprises: 10 * PCR damping fluid, MgCl
2, dNTP, Taq archaeal dna polymerase, BSA, hemophilus influenzae positive control dna, 3 pairs of hemophilus influenzae primers; It is characterized in that said 3 pairs of hemophilus influenzae primers are following:
The 1st pair: 5 '-GAAATGGTGCTGCTCAAACTTT-3 ', with 5 '-TGCGATGTTGTATTCTGGTGTA-3 ';
The 2nd pair: 5 '-CGATCAAGAGCCACATTTAAGC-3 ' and 5 '-CTTCTGAGCAATAGCCAACGA-3 ';
The 3rd pair: 5 '-CGTATTATCGGAAGATGAAAGTGC-3 ' and 5 '-AGGATGTCAAGAGTAGGTAAGGTT-3 '.
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| CN114790489A (en) * | 2021-11-04 | 2022-07-26 | 江汉大学 | MNP (MNP) marker site of haemophilus influenzae, primer composition, kit and application of MNP marker site |
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| CN101545010A (en) * | 2009-05-11 | 2009-09-30 | 北京海康基因芯片开发有限公司 | Method for detecting infectious disease pathogens and kit |
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| CN101545010A (en) * | 2009-05-11 | 2009-09-30 | 北京海康基因芯片开发有限公司 | Method for detecting infectious disease pathogens and kit |
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| Title |
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| SHEREEN SHOMA等: "Rapid Detection of Haemophilus influenzae Type b in Bangladeshi Children with Pneumonia and Meningitis by PCR and Analysis of Antimicrobial Resistance", 《J HEALTH POPUL NUTR》 * |
| THIÊN-TRÍ LÂM等: "New diagnostic PCR for Haemophilus influenzae serotype e based on the cap locus of strain ATCC 8142", 《INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY》 * |
| XIN WANG等: "Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae", 《INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114790489A (en) * | 2021-11-04 | 2022-07-26 | 江汉大学 | MNP (MNP) marker site of haemophilus influenzae, primer composition, kit and application of MNP marker site |
| CN114790489B (en) * | 2021-11-04 | 2023-06-20 | 江汉大学 | MNP (MNP) marking site of haemophilus influenzae, primer composition, kit and application of MNP marking site |
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