De­vel­op­ment of ther­mal­ly switch­able dos­ing sys­tems for a LAMP-rapid de­tec­tion of MRSA

IGF 17775 N

Due to lack of hy­giene each year be­tween 0.8 and 1.2 mil­lions of hos­pi­tal-ac­quired in­fec­tions occur in Ger­many, of which 20,000 to 70,000 are fatal. Me­thi­cillin-re­sis­tant Staphy­lo­coc­cus au­reus (MRSA) now rep­re­sent a 12.5 % share of one of the most com­mon caus­es of noso­co­mi­al in­fec­tions. Al­ready the es­tab­lish­ment and con­trol of hy­giene pro­tec­tion mea­sures lead to an ef­fi­cient pre­ven­tion of MRSA in­fec­tions. There­fore, reg­u­lar proof of suc­cess­ful­ly per­formed dis­in­fec­tion mea­sures by hos­pi­tal op­er­a­tors and clean­ing ser­vices is of in­creas­ing im­por­tance.

Since suit­able rapid meth­ods for self-mon­i­tor­ing are not avail­able, clean­ing ser­vice providers cur­rent­ly use mi­cro­bi­o­log­i­cal meth­ods like con­tact plates or swabs for sam­pling sur­faces. Af­ter­wards the con­tact plates must be in­cu­bat­ed and eval­u­at­ed by ex­ter­nal mi­cro­bi­o­log­i­cal lab­o­ra­to­ries. This method is cost-in­ten­sive and re­quires at least 3 days be­fore re­sults are avail­able.

The aim of the re­search pro­ject was the de­vel­op­ment of a rapid MRSA de­tec­tion method for sur­faces, which can be eas­i­ly used by clean­ing ser­vices as self-con­trol. Be­cause of this, ev­i­dence of suc­cess­ful­ly per­formed clean­ing and dis­in­fec­tion mea­sures are avail­able at lat­est after 3 hours so that a con­tin­u­ous con­trol and doc­u­men­ta­tion with­in self-con­trols will be made pos­si­ble.

As basis for the de­vel­op­ment of the rapid test sys­tem, LAMP-PCR for the spe­cif­ic ge­net­ic de­ter­mi­na­tion of MRSA was es­tab­lished. The am­pli­fied DNA-amounts can be de­ter­mined se­mi-quan­ti­ta­tive­ly due to the de­vel­oped colour ref­er­ence scale. To ex­clude false-pos­i­tive re­sults, an ini­tial step for the degra­da­tion of ex­tra­cel­lu­lar DNA from dead mi­croor­gan­isms was de­vel­oped. In order to sub­se­quent­ly re­lease the need­ed tar­get DNA from MRSA, op­ti­mal lysis and DNA ex­trac­tion con­di­tions were de­ter­mined. For the re­moval of in­hibit­ing ef­fects of the used lysis reagents, an ad­di­tion­al DNA-clean­ing step based on the use of DNA-bind­ing mag­net­ic beads was de­vel­oped.

For sub­sti­tu­tion of the need­ed pipet­ting steps for the LAMP-sys­tem, hy­dro­gels, which re­leased de­fined vol­umes of lysis as well as LAMP reagents at spe­cif­ic tem­per­a­tures, were de­vel­oped as ther­mal­ly switch­able dos­ing sys­tems. The ap­pli­ca­bil­i­ty of the ther­mal­ly switch­able dos­ing sys­tems was anal­ysed with re­gard to their ther­mosen­si­tive swelling prop­er­ties, me­chan­i­cal sta­bil­i­ty as well as load­ing and un­load­ing prop­er­ties. Based on these re­sults, a func­tion­al model of the LAMP-rapid test sys­tem for MRSA de­tec­tion in com­bi­na­tion with ther­mal­ly switch­able dos­ing sys­tems was de­vel­oped.

Thus, a rapid MRSA de­tec­tion method was de­vel­oped that can be used as part of on-site self-mon­i­tor­ing with­in qual­i­ty man­age­ment sys­tems, in order to prompt­ly de­tect pos­si­ble fail­ures in the dis­in­fec­tion pro­cess and, if nec­es­sary, im­me­di­ate­ly ini­ti­ate cor­rec­tive ac­tions.

The research report is available on request from FRT.

The IGF-pro­ject IGF 17775 N of the re­search as­so­ci­a­tion Europäische Forschungs­ge­mein­schaft Reini­gungs- und Hy­gien­etech­nolo­gie e.V., Cam­pus Ficht­en­hain 11, 47807 Krefeld, was sup­port­ed with­in the fund­ing pro­gram „In­dus­trielle Gemein­schafts­forschung und -en­twick­lung (IGF)“ by the Fed­er­al Min­istry of Eco­nom­ic Af­fairs and Cli­mate Ac­tion due to a de­ci­sion of the Ger­man Par­lia­ment.