MICROORGANISM CONCENTRATION METHOD WITH ELASTIC POLYMERS

Abstract

Identification of infectious agents, in samples like blood, urine, mouthwash obtained from patients, is the most important tool for laboratory diagnosis of infectious diseases. Due to their technical nature, diagnostic tests can use only a small part of the sample obtained from the patient. For that reason, it is very important to concentrate infectious agents into a small volume of sample that will be used in diagnostic tests, to increase their sensitivity. Additionally, there may be substances that interfere by working of the diagnostic tests based on nucleic acid amplification like polymerase chain reaction (PCR). It is important to remove these substances so that this kind of diagnostic tests can work properly. This invention is a method of concentrating infectious agents in biological samples by using elastic polymer meshes. When added to liquid biological samples, these meshes remove water and small molecules from their environment, diminish the volume of the sample and thus enable concentrating the microorganisms Concentration of microorganism and removal of substances that inhibit the working of diagnostic methods, increase the sensitivity of these methods.

Claims

1- It is a method, that reduces the volume of fluid biological samples by removing water and other small molecules with molecular weight less than 500 Daltons, with elastic hydrophilic polymer meshes, thereby increasing the sensitivity of the diagnostic tests used in the detection of infectious agents and kits that depend on this method.

2- The “elastic polymer meshes” used in the method defined in claim 1, are characterized in that they are made of polyacrylamide or polyacrylic acid or polymethacrylate and its derivatives or dextran or agar or agarose or gelatin or glycogen or all kinds of other hydrophilic polymers.

3- The “diagnostic tests” used in the method defined in claim 1, are either microscopic examination, or culture or antigen and antibody detection by immunological tests including immunochromatographic tests and Enzyme Linked Immunosorbent Assay (ELISA), or nucleic acid amplification tests including polymerase chain reaction (PCR), loop-mediated amplification (LAMP), strand displacement amplification (SDA) and rolling circle amplification that are used in detection infectious agents.

Description

EXAMPLES

[0014] 1—Suspensions of bacteria, Escherichia coli, Staphylococcus aureus, Candida albicans and Mycobacterium tuberculosis H37Ra, were prepared in sterile 0.9% NaCl solution from fresh cultures. 10 ml of the suspensions were placed in a sterile tube, and previously synthesized and dried elastic polymers were added into the suspensions and waited for 5 minutes for concentration. 1/10 serial dilutions were made by using 100 microliters of unconcentrated and concentrated liquids, again using 0.9% NaCl. Then, 10 microliters of Escherichia coli and Staphylococcus aureus dilutions were inoculated to Mueller Hinton Agar, dilutions of Candida albicans to Sabouraud Dextrose Agar and Mycobacterium tuberculosis H37Ra dilutions to Löwenstein-Jensen media. Mueller Hinton and Sabouraud Dextrose Agar media were kept for 2 days and Löwenstein-Jensen media for 3 weeks in a 37° C. incubator. At the end of these periods, the number of colony forming units per milliliter of bacterial suspensions, were determined by counting the colonies formed in the media. An increase of 20 to 40-fold in samples concentrated by elastic polymers, relative to the dilute suspensions of all microorganisms tested was determined.

[0015] 2—Samples that will be tested were prepared by adding SARS-CoV2 (new coronavirus) which were grown in Vero cells and than inactivated, into viral transport medium. In these samples, SARS-CoV2 RNA was investigated with Real-Time PCR before and after concentration by elastic polymers after extraction of RNA. It was identified that in samples concentrated with the polymer, SARS-CoV2 RNA was detected 4 to 6 cycles earlier, which indicated that the amount of virus in these samples was increased by 16 to 64 times.

[0016] We can conclude that this new method of concentration of microorganism including viruses with the elastic polymer meshes, has several advantages over previously described methods such as being a rapid, easy and effective method.