CN1865451A - PCR detection reagent kit for sexual disease mixed infection - Google Patents

Abstract

The invention discloses a synchronizing detection PCR agent box (hepatitis B virus, human immunodeficiency virus, human breast tumor virus, lues helicoid, human cytomegalovirus, simple herpesvirus, white oidiomycin, urea-dissolving mycoplasma, gonotoxin and Chlamydi trachomatis), which consists of blood type and secretion stains type. The user adds predisposed sample in the enlarging pipe to start enlarging reaction, which finishes detecting work simply and rapidly. The invention can detect ten venereal disease causal agents, which is fit for customs quarantine, prevailing monitor and hospital screen examination.

Description

PCR detection reagent kit for sexual disease mixed infection

Technical field

The present invention relates to biological medicine technology, particularly the polymerase chain reaction test kit of synchronous detection sexual disease mixed infection is called for short PCR detection reagent kit for sexual disease mixed infection.

Background technology

Sexually transmitted disease (STD) (venereal disease) is that a class mainly contacts the disease of propagating by infecting with the property between the infected not.Become one of the fastest transmissible disease of ascensional range in China's venereal disease, the kind of the venereal disease pathogenic agent of the sickness rate of venereal disease, discovery and the problem relevant with other transmissible diseases constantly increase, and spreading of AIDS speed has also significantly been accelerated in taking place frequently of venereal disease.Found at present can through the pathogenic agent of sex track transmission by original 4 kinds expand to now 20 surplus kind, comprise 7 big classes such as bacterium, fungi, virus, chlamydozoan, mycoplasma, spirochete, parasite.According to estimates, nearly 50% sexually infected person is asymptomatic, if delay in diagnosis or treat and untimelyly not only can become important contagium is quickened the propagation of venereal disease, and can cause the generation of complication and sequela, cause chronic inflammatory diseases, infertile even canceration etc.Along with increasing of venereal disease, the problem of polyinfection is also more and more serious, and polyinfection is relatively more typical with a kind of venereal disease clinical manifestation and symptom usually, and the concealment of the symptom of other one or more venereal diseases very easily causes to fail to pinpoint a disease in diagnosis Lou and controls.

The propagation of taking strong behavior intervention measure active prevention pathogenic agent is that the blocking-up transmissible disease takes place and the popular effective means, and screen the pathogenic agent carrier rapidly and accurately is the prerequisite of implementing effectively prevention and treatment, for reduce venereal disease especially acquired immune deficiency syndrome (AIDS) have positive effect at China popular.Traditionally, venereal disease etiological diagnosis method comprises culture method and non-culture method.Culture method sensitivity, special is the standard method of some screening for venereal disease, but culture method length consuming time, the collection of sample is all had higher requirement and some pathogenic agent is difficult to or still can not carry out vitro culture (as human papillomavirus) with transporting.Non-culture method (as smear for microscopic examination, serological test etc.) is convenient, but susceptibility is low, and not obvious or symptomless infection person is difficult to detect for symptom.Polymerase chain reaction (PCR) technology combines the advantage of culture method and non-culture method: have higher sensitivity and specificity, can infect the lower detections of pathogenic agent carrying capacity such as early stage or symptomless infection; Collection of specimens is convenient, can use Noninvasive sample (vagina or vulva sample, the urine etc. of asking for as the patient); Less demanding to the sample shipping conditions; Detect fast.The domestic PGR detection kit that has developed is a sick box at present, is unfavorable for the examination of polyinfection venereal disease.

Summary of the invention

Purpose of the present invention be intended to overcome the deficiencies in the prior art and provide that a kind of detection is quick, less demanding to the sample shipping conditions, collection of specimens is convenient, have more hypersensitivity and specificity, multiple STD pathogenic agent is carried out the PCR detection reagent kit for sexual disease mixed infection of synchronous detection.

The object of the present invention is achieved like this:

A kind of PCR detection reagent kit for sexual disease mixed infection, this detection kit comprises pretreatment fluid, amplification pipe and positive template, and described positive template contains hepatitis B virus, human immunodeficiency virus, human papillomavirus, treponema pallidum, human cytomegalic inclusion disease virus, hsv or wherein arbitrary combination or single plasmid vector of planting the specific gene sequence of pathogenic agent at blood type amplification Guan Zhongwei; Contain Candida albicans, Ureaplasma urealyticum, gonococcus, chlamydia trachomatis or wherein arbitrary combination or the singly plasmid vector of the specific gene sequence of kind pathogenic agent at juice type amplification Guan Zhongwei.

---described pretreatment fluid is the 0.1-1.0mol/L sodium hydroxide solution.

---described amplification pipe is divided into blood type or/and the juice type.

---described amplification pipe is made up of archaeal dna polymerase, 4 kinds of deoxynucleoside triphosphates, damping fluid, magnesium ion, mix primer and water.

---the specific gene sequence of described plasmid vector is following listed gene or arbitrary combination wherein: the hepatitis B virus S gene of blood type, human immunodeficiency virus gag gene, the main agricultural glutelin L1 gene of human papillomavirus, treponema pallidum TP0574 gene, human cytomegalic inclusion disease virus UL123 gene, hsv UL30 gene; The Candida albicans autonomously replicating sequence of juice type, Ureaplasma urealyticum urase complex body ureB gene ureC gene, gonococcus B protein gene, chlamydia trachomatis DRP3 gene.

---mix primer in the described amplification pipe in blood type at hepatitis B virus, human immunodeficiency virus, human papillomavirus, treponema pallidum, human cytomegalic inclusion disease virus, hsv or the primer that designs of arbitrary combination or single distinguished sequence of planting pathogenic agent wherein; In the juice type at Candida albicans, Ureaplasma urealyticum, gonococcus, chlamydia trachomatis or the primer that designs of arbitrary combination or single distinguished sequence of planting pathogenic agent wherein.

---described mix primer is following listed Nucleotide or contains wherein nucleotide sequence that N represents 4 kinds of any deoxynucleoside triphosphates;

(1) blood type

Hepatitis B virus: 5 '-NNNNNCCTGCTGGTGGCTCCAGTTCCGGANNNNN-3 '

5’-NNNNNTAGGGTTTAAATGTATACCCAAAGNNNNN-3’

Human immunodeficiency virus: 5 '-NNNNNAGAGGATCCAGGGGCAAATGGTANNNNN-3 '

5’-NNNNNTCCCGGTACCTGTCATGCTGTCANNNNN-3’

Human papillomavirus: 5 '-NNNNNGTGGGGGAACCTGTGCCTGATNNNNN-3 '

5’-NNNNNTGTACCATTTGGGGGAGGCGANNNNN-3’

Treponema pallidum: 5 '-NNNNNTTAGTAACGCTTGGGTCAGTCNNNNN-3 '

5’-NNNNNTTGTTGGTTGTAGGCTGTGGCNNNNN-3’

Human cytomegalic inclusion disease virus: 5 '-NNNNNAGCCATTGGTGGTCTTAGGGANNNNN-3 '

5’-NNNNNTTGGGCTAACTATGCAGAGCANNNNN-3’

Hsv: 5 '-NNNNNCCACCTGACGGTGTATTACAANNNNN-3 '

5’-NNNNNGCGCGACCGTCTCCTCTACCTNNNNN-3’

(2) juice type

Candida albicans: 5 '-NNNNNTAGCGATGAGGTAGTGCAAGNNNNN-3 '

5’-NNNNNGCTGCAGCTACGATTGTTAGNNNNN-3’

Ureaplasma urealyticum: 5 '-NNNNNTCCAGGTAAATTAGTACCAGGNNNNN-3 '

5’-NNNNNTCTCCTAATCTAACGCTATCANNNNN-3’

Gonococcus: 5 '-NNNNNGCTACGCATACCCGCGTTGCTNNNNN-3 '

5’-NNNNNCGAAGACCTTCGAGCAGACATNNNNN-3’

Chlamydia trachomatis: 5 '-NNNNNAGCACTCCAATTTCTGACTGTNNNNN-3 '

5’-NNNNNTCGATGATTTGAGCGTGTGTANNNNN-3’

The present invention is a kind of to 10 kinds of venereal disease pathogenic agent---hepatitis B virus (HBV), human immunodeficiency virus (HIV-1) they are hiv virus, human papillomavirus (HPV), treponema pallidum (TP), Human cytomegalic inclusion disease virus (HCMV), cook the associating PCR detection kit that exanthema virus (HSV), Candida albicans (CA), Ureaplasma urealyticum (UU), gonococcus (NG), chlamydia trachomatis (CT) are realized synchronous detection merely, and whole detection step comprises sample pretreatment-amplification-electrophoresis detection result.The amplification pipe of test kit is divided into blood type and (detects hepatitis B virus, the human immunodeficiency virus, human papillomavirus, treponema pallidum, human cytomegalic inclusion disease virus, hsv) and the juice type (detect Candida albicans, Ureaplasma urealyticum, gonococcus, chlamydia trachomatis) two kind, 10 kinds of venereal disease pathogen gene primers and the required reagent of PCR reaction system are added in the amplification pipe in advance by blood type and juice type, one people's portion, need not packing, the user only needs that pretreated sample is added amplification pipe startup amplified reaction and gets final product, thereby avoid the crossed contamination that may occur in the operating process, finish testing quickly and easily.This test kit carries out synchronous detection to diversity disease pathogen body, is fit to the examination of sexual disease mixed infection, and single this primary first-order equation of increment can detect multiple pathogenic agent, and it is still incomplete to have overcome present domestic venereal disease detection kit, the disadvantage of a sick box.Test kit susceptibility height is fit to infect early stage and symptomless infection person's detection.That test kit uses is simple, detect fast, all has higher use value in a big way infection population or high risk population's generaI investigation and the detection of contagium.

Primer in the test kit is at the distinguished sequence of every kind of pathogenic agent and design, the amplified production high specificity, and 10 kinds of pathogenic agent amplified production molecular weight sizes change in gradient, and are clear and legible.Test kit also is provided with FFI positive template, the amplified production control test, and the result comes into plain view.

Description of drawings

Fig. 2 is the increase distribution tabulation of pipe of the present invention.

Fig. 1 is the PCR product analysis figure of venereal disease sample of the present invention.

In Fig. 1: the positive molecular weight standard of M; 1-4 is different sample amplified productions.

Embodiment

Embodiment 1: the design of primer sequence

1) hepatitis B virus (hepatitis B virus): S (HBsAg) gene (155bp-835bp) and upstream and downstream partial sequence: 56bp-841bp thereof, fragment length 786bp.

Upstream primer: 5 '-CCTGCTGGTGGCTCCAGTTCCGGA-3 '

Downstream primer: 5 '-TAGGGTTTAAATGTATACCCAAAG-3 '

2) human immunodeficiency virus (human immunodeficiency virus type 1): gag gene (790bp-2295bp): 1216bp-1841bp, fragment length 626bp.

Upstream primer: 5 '-AGAGGATCCAGGGGCAAATGGTA-3 '

Downstream primer: 5 '-TCCCGGTACCTGTCATGCTGTCA-3 '

3) Candida albicans (Candida albicans): autonomously replicating sequence: 27bp-710bp, fragment length 684bp.

Upstream primer: 5 '-TAGCGATGAGGTAGTGCAAG-3 '

Downstream primer: 5 '-GCTGCAGCTACGATTGTTAG-3 '

4) Ureaplasma urealyticum (Ureaplasma urealyticum): urase complex body ureB gene (495-869bp) and ureC gene (908bp-2704bp): 521bp-981bp, fragment length 461bp.

Upstream primer: 5 '-TCCAGGTAAATTAGTACCAGG-3 '

Downstream primer: 5 '-TCTCCTAATCTAACGCTATCA-3 '

5) human papillomavirus (human papillomavirus type 6b): main capsid protein L 1 gene (5789bp-7291bp) 6575bp-7012bp, fragment length 438bp.

Upstream primer: 5 '-GTGGGGGAACCTGTGCCTGAT-3 '

Downstream primer: 5 '-TGTACCATTTGGGGGAGGCGA-3 '

6) gonococcus (Neisseria gonorrheae): pJD1 plasmid; B protein gene (2912bp-3553bp): 3141bp-3531bp, fragment length 391bp.

Upstream primer: 5 '-GCTACGCATACCCGCGTTGCT-3 '

Downstream primer: 5 '-CGAAGACCTTCGAGCAGACAT-3 '

7) treponema pallidum (Treponema pallidum): TP0574 gene (12870bp-14174bp): 13810bp-14132bp, fragment length 323bp.

Upstream primer: 5 '-TTAGTAACGCTTGGGTCAGTC-3 '

Downstream primer: 5 '-TTGTTGGTTGTAGGCTGTGGC-3 '

8) human cytomegalic inclusion disease virus (human cytomegalovirus): UL123 gene extron 4 (171006bp-172225bp): 171800bp-172045bp, fragment length 246bp.

Upstream primer: 5 '-AGCCATTGGTGGTCTTAGGGA-3 '

Downstream primer: 5 '-TTGGGCTAACTATGCAGAGCA-3 '

9) chlamydia trachomatis (Chlamydia trachomatis): pLGV440 cryptic plasmid; DRP3 gene (2225bp-3580bp): 2544bp-2763bp, fragment length 220bp.

Upstream primer: 5 '-AGCACTCCAATTTCTGACTGT-3 '

Downstream primer: 5 '-TCGATGATTTGAGCGTGTGTA-3 '

10) hsv (herpes simplex virus 2): UL30 (archaeal dna polymerase) gene (63265bp-66987bp): 66429bp-66544bp, fragment length 116bp.

Upstream primer: 5 '-CCACCTGACGGTGTATTACAA-3 '

Downstream primer: 5 '-GCGCGACCGTCTCCTCTACCT-3 '

Embodiment 2: the component preparation of amplification pipe

The cumulative volume of amplified reaction mixed solution is 23 μ L, and it is formed as table 1.

Embodiment 3: sample pretreatment and gene amplification

Cotton swab is put into 500 μ l physiological saline stir, with the centrifugal 3min of 12000r/min, abandon supernatant then, precipitation adds 50 μ l pretreatment fluids, and mixing boils 10min, the centrifugal 5min of 12000r/min gets 2 μ L supernatants and is added in the amplified reaction pipe, starts amplified reaction.Reaction conditions is as follows: 93 ℃ of sex change in 3 minutes; 93 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ of totally 35 circulations in 1 minute; 72 ℃ of extensions in 5 minutes.

Embodiment 4: the result detects

Directly from reacted amplification pipe, get 10 μ L application of samples,, on ultraviolet device, observe, contrast positive molecular weight standard containing electrophoresis on 1% sepharose of ethidium bromide (5V/cm, 40 minutes), if occur band on the corresponding position, as Fig. 1 then for this pathogenic agent positive:

Sample 3 does not detect pathogenic infection as seen from Figure 1; Sample 1 amplified production stripe size is 220bp, is the single infection of chlamydia trachomatis (CT); Sample 2 and 4 two amplified production stripe size are respectively 391 and 220bp, are gonococcus (NG) and chlamydia trachomatis double infection.

Claims (7)

1. PCR detection reagent kit for sexual disease mixed infection, it is characterized in that this detection kit comprises pretreatment fluid, amplification pipe and positive template, described positive template contains hepatitis B virus, human immunodeficiency virus, human papillomavirus, treponema pallidum, human cytomegalic inclusion disease virus, hsv or wherein arbitrary combination or single plasmid vector of planting the specific gene sequence of pathogenic agent at blood type amplification Guan Zhongwei; Contain Candida albicans, Ureaplasma urealyticum, gonococcus, chlamydia trachomatis or wherein arbitrary combination or the singly plasmid vector of the specific gene sequence of kind pathogenic agent at juice type amplification Guan Zhongwei.

2. detection kit according to claim 1 is characterized in that described pretreatment fluid is the 0.1-1.0mol/L sodium hydroxide solution.

3. detection kit according to claim 1 is characterized in that described amplification pipe is divided into blood type or/and the juice type.

4. detection kit according to claim 1 is characterized in that described amplification pipe is made up of archaeal dna polymerase, 4 kinds of deoxynucleoside triphosphates, damping fluid, magnesium ion, mix primer and water.

5. detection kit according to claim 1, the specific gene sequence that it is characterized in that described plasmid vector are following listed gene or arbitrary combination wherein: the hepatitis B virus S gene of blood type, human immunodeficiency virus gag gene, the main capsid protein L 1 gene of human papillomavirus, treponema pallidum TP0574 gene, human cytomegalic inclusion disease virus UL123 gene, hsv UL30 gene; The Candida albicans autonomously replicating sequence of juice type, Ureaplasma urealyticum urase complex body ureB gene ureC gene, gonococcus B protein gene, chlamydia trachomatis DRP3 gene.

6. detection kit according to claim 1, the mix primer in the pipe that it is characterized in that increasing in blood type at hepatitis B virus, human immunodeficiency virus, human papillomavirus, treponema pallidum, human cytomegalic inclusion disease virus, hsv or the primer that designs of arbitrary combination or single distinguished sequence of planting pathogenic agent wherein; In the juice type at Candida albicans, Ureaplasma urealyticum, gonococcus, chlamydia trachomatis or the primer that designs of arbitrary combination or single distinguished sequence of planting pathogenic agent wherein.

7. according to claim 4 and 6 described detection kit, it is characterized in that mix primer is following listed Nucleotide or contains wherein nucleotide sequence, N represents 4 kinds of any deoxynucleoside triphosphates;

(1) blood type

Hepatitis B virus: 5 '-NNNNNCCTGCTGGTGGCTCCAGTTCCGGANNNNN-3 '

5’-NNNNNTAGGGTTTAAATGTATACCCAAAGNNNNN-3’

Human immunodeficiency virus: 5 '-NNNNNAGAGGATCCAGGGGCAAATGGTANNNNN-3 '

5’-NNNNNTCCCGGTACCTGTCATGCTGTCANNNNN-3’

Human papillomavirus: 5 '-NNNNNGTGGGGGAACCTGTGCCTGATNNNNN-3 '

5’-NNNNNTGTACCATTTGGGGGAGGCGANNNNN-3’

Treponema pallidum: 5 '-NNNNNTTAGTAACGCTTGGGTCAGTCNNNNN-3 '

5’-NNNNNTTGTTGGTTGTAGGCTGTGGCNNNNN-3’

Human cytomegalic inclusion disease virus: 5 '-NNNNNAGCCATTGGTGGTCTTAGGGANNNNN-3 '

5’-NNNNNTTGGGCTAACTATGCAGAGCANNNNN-3’

Hsv: 5 '-NNNNNCCACCTGACGGTGTATTACAANNNNN-3 '

5’-NNNNNGCGCGACCGTCTCCTCTACCTNNNNN-3’

(2) juice type

Candida albicans: 5 '-NNNNNTAGCGATGAGGTAGTGCAAGNNNNN-3 '

5’-NNNNNGCTGCAGCTACGATTGTTAGNNNNN-3’

Ureaplasma urealyticum: 5 '-NNNNNTCCAGGTAAATTAGTACCAGGNNNNN-3 '

5’-NNNNNTCTCCTAATCTAACGCTATCANNNNN-3’

Gonococcus: 5 '-NNNNNGCTACGCATACCCGCGTTGCTNNNNN-3 '

5’-NNNNNCGAAGACCTTCGAGCAGACATNNNNN-3’

Chlamydia trachomatis: 5 '-NNNNNAGCACTCCAATTTCTGACTGTNNNNN-3 '

5’-NNNNNTCGATGATTTGAGCGTGTGTANNNNN-3’

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