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Method for recovering microbial cells

A technology of microbial cells and microorganisms, which is applied in the methods of sampling biological materials, biochemical equipment and methods, and the determination/inspection of microorganisms. Effect

Pending Publication Date: 2019-06-14
Q LINEA AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other methods utilizing harsh alkaline conditions are also known in the art (Banada et al. 2012. PLoS One 7, e31126), but these methods do not recover viable microbial cells; lysis of microbial cells is known to occur at higher pH values ​​( e.g. pH11 and above)

Method used

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  • Method for recovering microbial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1 - Addition of protease-containing buffer to sample to enhance its filterability

[0111] Initial experiments were performed to determine the effect of adding protease-containing buffers on the filterability of the samples. Samples were prepared using E. coli as described above and incubated with buffered solutions containing proteinase K at a range of different pH values. For comparison, additional samples were incubated with proteinase K-free buffer solution at pH 10.5. The results show that the filterability of each sample after treatment with each buffer solution is shown in Table 1.

[0112] Table 1.

[0113] sample

pH 9

pH 9.5

pH 10

pH 10.5

pH 11

pH 11.5

buffer only

-

-

-

<1ml

-

-

Buffer+Protease K

15ml

15ml

15ml

15ml

15ml

15ml

Recovery of live cells after backwashing

5％

5％

20％

5％

0％

0％

Viability of cells after incubation (without filtr...

Embodiment 2

[0118] Example 2 - Vitality of microbial cells

[0119] Samples were prepared using E. coli (Gram-negative bacteria) and S. pyogenes (Gram-positive bacteria) as described above. Samples were contacted with pH 10.0 buffer containing proteinase K, then washed with PBS, subsequently treated with DNaseI for 5 minutes, and the cells on the filter resuspended as described above. The microbial cells recovered from the samples were subsequently tested for viability and the results are shown in Table 3.

[0120] table 3.

[0121]

[0122] Both Gram-negative E. coli and Gram-positive S. pyogenes were found to be viable at pH 10.0, with S. pyogenes appearing to have higher viability under these conditions.

[0123] The recovered cells were used to prepare bacterial DNA using a DNA kit from Molzym (Molzym GmbH, Bremen, Germany) and the results were quantified by real-time PCR to confirm the presence of bacterial as well as residual human DNA.

[0124] The approximate tota...

Embodiment 4-p

[0133] Example 4 - Effect of pH on Filterability and Viability

[0134] Multiple CAPS buffers were prepared and adjusted to pH 7, 8, 9, 9.5, 10, 10.5 and 11 each at 0.3M. Samples were added to each CAPS buffer and incubated with proteinase K. Samples were then filtered using polyamide filters and resuspended using phosphate buffered saline (PBS) solution. Recovery was calculated based on colony formation (ie only viable cells were counted). Filtration and recovery of microbial cells at different pH values ​​are shown in Table 6.

[0135] Table 6.

[0136]

[0137] Improvement in filterability was observed in buffer treatments at pH 11-11.5, however, microbial viability was impaired at these high pH values. After finding that buffers with a pH of at least pH 9 can be used to enhance the filterability of clinical samples, the effect of buffers with a wider pH range on enhancing the filterability of clinical samples was tested. The results are shown in Table 7.

[01...

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Abstract

The present invention provides a method of recovering viable microbial cells from a complex sample, said method comprising: a) providing a sample having a volume of at least 1 ml; b) contacting said sample with a buffer solution and one or more proteases, wherein said buffer solution has a pH of at least pH 6 and less than pH 11, wherein said buffer solution and said one more proteases do not comprise a detergent or a chaotrope, and wherein the buffer solution / protease / sample mixture is non-hypotonic; c) filtering the mixture obtained in step (b) through a filter suitable for retaining microbial cells; and d)recovering the microbial cells retained by the filter in step (c), wherein the recovered microbial cells are viable, and a microbial recovery device for the same.

Description

technical field [0001] The present invention relates to methods for the recovery of viable microorganisms (microbial cells) from large quantities of complex samples. In particular, the sample may be a clinical sample or may comprise a clinical sample, especially blood, such as a blood sample in a blood culture bottle. The present invention is based on the surprising discovery that the addition of certain buffer solutions, especially simple buffer solutions containing proteases, enhances the filterability of complex samples. This allows rapid and efficient recovery of viable microbial cells from complex samples for subsequent identification and biochemical testing. Background technique [0002] Microbial infections represent a major class of human and animal diseases of great clinical and economic importance. Although various classes and types of antimicrobial agents are available to treat and / or prevent microbial infections, antimicrobial resistance is a huge and growing p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/24C12N1/20
CPCC12Q1/24C12N1/20C12M33/14C12M21/18G01N1/4005G01N1/4077G01N2001/4016G01N2001/4088
Inventor 马库斯·克林特斯特哈勒尔·奥斯曼马茨·格尔伯格
Owner Q LINEA AB
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