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Primer composition for detecting legionella and kit using the same

A primer combination and kit technology, applied in the field of molecular biology detection of Legionella, can solve the problems of specificity dependence, long incubation time, unfavorable rapid detection and diagnosis of Legionella, and achieve the effect of improving sensitivity and simple method.

Active Publication Date: 2014-11-05
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many problems: firstly, Legionella culture requires strict and long culture time, and it is difficult to isolate from clinical and environmental specimens due to the overgrowth of other miscellaneous bacteria
It has been reported that the sensitivity of culturing Legionella from respiratory secretions is 30-70%. In addition, in some specimens, Legionella in a viable non-cultivable state is present. Therefore, culture alone is not suitable for detection of clinical and environmental specimens. Facilitate the rapid detection and diagnosis of Legionella
Secondly, although direct fluorescent antibody (DFA) detection is fast, the sensitivity is low, about 25-75%, and the specificity depends on the experience of the staff
Third, when the indirect fluorescent antibody (IFA) or agglutination test is used to detect the antibody, since the patient's antibody level can only be detected one week after infection with the disease, it is not suitable for early diagnosis, and the detection sensitivity is about 75%.
Amplification of 870bp gene fragment can only detect Legionella pneumophila

Method used

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  • Primer composition for detecting legionella and kit using the same
  • Primer composition for detecting legionella and kit using the same
  • Primer composition for detecting legionella and kit using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 PCR primer combination for detecting Legionella

[0034] The primer combination includes primer pair I for PCR specific amplification of mip gene and primer pair II for PCR specific amplification of 16S rRNA. The primer sequence is as follows:

[0035] Primer pair I:

[0036] Upstream primer QT1 5′-GCAATGGCTAAAGGCATG-3′

[0037] Downstream primer QT2 5′-AATACAACAACGCCTGGCTTG-3′

[0038] Primer pair II:

[0039] Upstream primer 16sQ-F 5′-AGGGTTGATAGGTTAAGAGC-3′

[0040] Downstream primer 16sQ-R 5′-CCAACAGCTAGTTGACATCG-3′

Embodiment 2

[0041] Example 2 Specificity and sensitivity analysis of PCR primer combination for detecting Legionella

[0042] The following PCR reaction system and reaction conditions were used to detect the specificity and sensitivity of the PCR primer combination of Example 1.

[0043] The PCR reaction system is calculated as 30μl:

[0044]

[0045]

[0046] The PCR reaction conditions are: 94°C for 2 minutes, 61°C for 45 seconds, 72°C for 45 seconds, 1 cycle; 94°C for 45 seconds, 61°C for 45 seconds, 72°C for 45 seconds, a total of 33 cycles.

[0047] 1. Perform double PCR amplification on different concentrations of Legionella pneumophila (Lp1) DNA to test the sensitivity of the method. The result is figure 1 As shown, two clearly visible bands (206bp and 386bp) are produced, and the minimum detection limit is 2×10 1 cfu / ml.

[0048] 2. Double PCR amplification of the international reference strain of Legionella pneumophila serotype 1-14 (Table 1), the results are as follows figure 2 As shown...

Embodiment 3

[0055] Example 3 Detection of Legionella by double PCR

[0056] Using the primer combination of Example 1 and the PCR reaction system and reaction conditions of Example 2, the 7 strains of Legionella isolated from the environment and the Legionella strains isolated from clinical patients were subjected to double PCR amplification. The result is Figure 5 As shown, both 206bp and 386bp fragments were amplified.

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Abstract

The invention provides a primer composition for detecting legionella in clinical and environmental specimens. The primer composition comprises a pair of primers (shown in the Seq ID No.1-2) for PCR specific amplification of a gene mip and a pair of primers (shown in the Seq ID No.3-4) for PCR specific amplification of 16S rRNA. The invention also provides a detection kit containing the primer composition. A test on 320 groups of phlegm and alveolar irrigation solutions of pneumonia patients and 210 groups of environmental water specimens proves that the primer composition has singularity of 100% and a lowest detection limit of 2*10<1> cfu / ml. The dual PCR system can be successfully used in bacterial strain identification and clinical and environmental specimen detection. The PCR detection method built based on the dual PCR system has the characteristics of simpleness, fastness and reliability and can be widely used in epidemiology preliminary investigation, environmental water monitoring and clinical specimen detection.

Description

Technical field [0001] The invention relates to the molecular biology detection of Legionella, in particular to a primer combination and a kit for detecting Legionella in clinical and environmental specimens. Background technique [0002] Legionella (Legionella) is a kind of bacteria that commonly exists in various artificial and natural water environments. It is the pathogen that causes Legionellosis (Legionellosis). It can be found in amoebas and human monocytes, mainly alveolar macrophages. Cells grow and multiply, causing pneumonia and Pontiac fever. Pneumonia usually has a rapid onset and high mortality. The onset of Pontiac fever is like a cold. The main symptoms are fever and the infection rate is high, but the prognosis is better than that of pneumonia, and there is no death. Epidemiological investigations have confirmed that Legionella is spread to people through the air through aerosols, often in cooling towers, hot water supply systems, steam concentrators, spa baths...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2537/143
Inventor 秦天任红宇邵祝军
Owner ICDC CHINA CDC
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