Primer combination for detecting PCR of Elizabethkingia meningoseptica

A technology of primer sequence and amplification primer, which is applied in the field of microbiology and molecular biology, and achieves the effects of wide application prospects, excellent specificity and sensitivity

Active Publication Date: 2017-08-22
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Regardless of the PCR or Real-time PCR method, the specific target gene (or nucleic acid fragment) is also used as the basis for clinical diagnosis. For clinical cases suspected of being infected by Elizabeth meningitidis, there is currently no target gene (or nucleic acid fragment) ) can be used for rapid PCR detection

Method used

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  • Primer combination for detecting PCR of Elizabethkingia meningoseptica
  • Primer combination for detecting PCR of Elizabethkingia meningoseptica
  • Primer combination for detecting PCR of Elizabethkingia meningoseptica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Establishment of PCR amplification system

[0077] Common PCR reagents and reaction systems and conditions:

[0078] Premix Taq Mix (TaKaRa lot: RR901A); primers were designed by Primer3.0 software and synthesized by Aoke Dingsheng; agarose (Biowest lot: 11176); GoldView nucleic acid stain (Solarbio lot: G8140); 100bp DNA Marker (full Formula gold lot: BM301-02); gel imaging system Bio-red GelDoc XR; SENSQUEST PCR amplifier; Eppendorf 5415D centrifuge; DYY-8C (Beijing Liuyi Instrument Factory).

[0079] reaction system:

[0080] name Volume (uL) Final concentration 2 x Taq Mix 25 upstream primer 2 400nM downstream primer 2 400nM template DNA 2 h 2 o

19 total capacity 50uL

[0081] Reaction conditions:

[0082]

[0083] SYBR Green Real-time PCR detection reagent and reaction system: UltraSYBR Mixture (Low ROX) (Kangwei Century lot: CW2601M); primers were designed by Primer3.0 software...

Embodiment 2

[0092] Embodiment 2: General PCR and SYBR Green Real-time PCR specificity verification:

[0093] Specific detection: Simultaneous detection of Elizabeth meningitidis (ATCC 13253), Neisseria meningitidis (CMCC29356), Haemophilus influenzae (CDC control isolate M5216), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC) 49619), Escherichia coli (ATCC 25922), Listeria monocytogenes (SLCC 2372), Mycoplasma pneumoniae (ATCC 29342), Bordetella pertussis (ATCC 9797), Klebsiella pneumoniae (CMCC 46114) and Mycobacterium tuberculosis ( H37Rv) was detected.

[0094] A, B, C, D, E, F, G, and H primer combinations were used to amplify and detect the common pathogenic bacteria that easily cause meningitis and upper respiratory tract symptoms above to verify the specificity of the primers.

[0095] The specificity detection results show that the primers of group A can produce amplification products of non-target fragment size against Haemophilus influenzae (the target fragm...

Embodiment 3

[0101] Embodiment 3: Ordinary PCR and SYBR Green Real-time PCR sensitivity verification

[0102] The nucleic acid at a concentration of 1 ng / uL of Elizabeth meningitidis (ATCC 13253) was used as the stock solution, and the diluted samples were tested by ordinary PCR and fluorescence quantitative PCR respectively.

[0103] Quantitative detection of DNA of freshly prepared Elizabeth meningitidis strain ATCC13253 and then diluted to 10 -1 ng / μL(1ng / μL≈4.28*10 5 copy / Ul), 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 - 6 ng / μL, 10 -7 ng / μL, 10 -8 ng / μL for sensitivity verification. Figure 12 Sensitivity results of A, B, C, D, E, F, G, and H primer combinations for the detection of Elizabeth meningitidis (ATCC 13253) by the common PCR method are shown. In the electrophoresis detection of each group, the lanes are from left to right. The order of arrangement is: 100bp DNA molecular weight marker, 10 -1 ng / μL template, 10 -2 ng / μL template, 10 - 3 ng / μL templa...

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Abstract

The invention discloses a primer sequence combination and a method for applying the primer sequence combination in PCR amplification. The amplification target in the method is specific fragments EMA inside the genome of Elizabethkingia meningoseptica, and the method shows excellent specificity and sensibility; and moreover, by using the method, the neisseria meningitidis, haemophilus influenzae, staphylococcus aureus, streptococcus pneumoniae, escherichia coli, listeria monocytogenes, mycoplasma pneumoniae, bordetella pertussis, klebsiella pneumoniae and mycobacterium tuberculosis and the other pathogenic bacteria which are likely to cause meningitis and diseases of the upper respiratory tract, and easy to be confused with the Elizabethkingia meningoseptica in an identification of bacteria, can be distinguished at one time. Additionally, the lower detection limit of the method can reach 10-4 ng per Mul.

Description

technical field [0001] The invention discloses a set of primer combinations for PCR, which belongs to the technical field of microorganisms and molecular biology. Background technique [0002] Meningoseptica Elizabeth was isolated from neonatal meningitis patients by Elizabeth O. King in 1959 and named Flavobacterium meningosepticum. In 1994, it was classified into the genus Chryseobacterium and named Chryseobacterium meningosepticum. In 2005, after 16S rRNA cluster analysis, A new genus was identified: Elizabethkingia, named Elizabethkingia meningoseptica. Elizabeth meningitidis can cause outbreaks of neonatal meningitis in premature infants and infants. [0003] Elizabeth meningitidis is a gram-negative bacillus that is widely distributed in natural environments (eg, freshwater, saltwater, and soil). Since the discovery in 1959 that Elizabeth meningitidis can cause neonatal meningitis, related outbreaks and clinical cases caused by this bacteria have been reported from t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2563/107
Inventor 高源邵祝军朱兵清徐丽
Owner ICDC CHINA CDC
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