Crispr-cas 12a combination to alleviate the false-positive in loop-mediated isothermal amplication-based diagnosis of Neisseria meningitidis

Loop mediated Isothermal amplicacation (LAMP) was recently suggested as a diagnostic tool for the identication of Neisseria meningitides. Howevere, this isothermal amplication is challenged by the fact its amplication leads to risks of obtaining false-positive results. Whereas, with abilities to accurately recognize specic sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen’s DNA targets complimerntary to its cognate RNA and acquires collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant uorescent-quencher-nucleic probes are present in the reaction, the non-specic cleavage of probes releases uorescences and establish diagnostic read-outs. In this study, we demonstrate a proof-of-concept that in relevant biochemical conditions, CRISPR/Cas12a and LAMP can work synchronously to identify genetics materials of Nesseria menitigistis at the level of 0.00004% in less than 2 h. Additionally, our clinical data also showed that the combinatory of CRISPR/Cas12a help to alleviate false positive result therefore enhance the specicity gained by the LAMP assays. real-time PCR in identication of patients infected with N. menitigitis: 51 CSF samples were equally subjected to conventional real-time PCR MetA genes as molecular target or LAMP assay or LAMP/Crispr cas12a combination. Data revealed that LAMP assay detected 18 positive cases, whereas LAMP/Crispr cas 12 identied only 13 cases that are fully matched to results acquired by real-time PCR.


Introduction
Neisseria meningitidis (NM) is a Gram-negative bacterium that causes severe meningitis and sepsis. These diseases require fast and accurate diagnostics to indicate proper antimicrobial therapies [1,2]. So far, together blood culture, polymerase chain reaction (PCR) is recommended as a routine technique for the diagnostic con rmation [3,4]. PCR requires laboratories with sophisticated infrastructures and well-trained personnel, therefore makes challenges for deploying in limited-resource areas. Loop-mediated isothermal ampli cation (LAMP) based approaches have been used to detect pathogens [5]. LAMP-based assays are faster and require no sophisticated instruments or/and skilled personnel, therefore have the advantage to use as on-site diagnostic device [6].
LAMP can gain PCR's sensitivity without complicated thermocycling, some LAMP assays can complete within 10 minutes. However, LAMP detection step acquired non-speci c indicators (such as Mg2+, intercalating dyes, labelled primers) that cannot distinguish spurious amplicons [7-9]. We documented several phenomena that real-time PCR protocols [4, 10] could not recapitulate positive results gained by LAMP reactions and some LAMP positive cases lacked meningitidis speci c clinical symptoms [1].
We suspected that LAMP assay might acquire given false positive potentials [7][8][9]. With abilities to accurately recognize speci c sequence, the CRISPR/Cas system holds promising potentials. In this system, the DNAse cas12a forms a complex with their cognate CRISPR RNAs to cleave pathogen's RNA or DNA in a sequence-speci c manner, afterwards, the collateral transcleavage activity is induced to cut unbiasedly the nearby off-target fragments. If relevant uorescent-quencher-nucleic probes are present in the reaction, the non-target cleavage of probes will release uorescent signals and establish diagnostics read-outs [11-13].

Results
We rst performed isothermal ampli cation assay using Bst DNA Polymerase with primers speci c for MetA gene of N. meningitidis. The reaction products were resolved against 1.5% agarose gel. It is impossible to distinguish the electrophoretic banding pattern between human DNA, E. coli DNA or N. meningitidis DNA (Fig. 1 upper-left panel). However, once these products were treated with Crispr-cas12a with gRNA sequence complementary to MetA gene of N. meningitidis, only uorescent signals from samples with N. meningitidis DNA was recorded. Thus, treatment of CRISPR-Cas12a helps alleviate falsepositive results by single-use of LAMP assay.
To evaluate the detection limit of LAMP/ CRISPR-Cas12a combination for the detection of N. meningitis DNA, we spiked series of 40, 400, 4,000 and 40,000 copies of N. meningitidis PCR amplicon into 25 mM Tris-EDTA pH 8 containing the background of 10 8 copies of E. coli PCR amplicon/ul (suppl. information).
These dilution points were used as the templates for Bst DNA Polymerase isothermal ampli cation at 55 o C for 45 minutes then treated with CRISPR cas12a (1.0 µM Cas12a per reaction), in the presence of 0,25 µM guide RNA and 0,25 µM uorescence labelled reporter at 55 o C for 30 minutes; the uorescent signal was recorded in 510 nm in Roche light cycler 480. The uorescence was detected at all prepared dilution points even at the lowest level of 4 × 10 1 copies (0.00004%) of N. meningitidis (Fig. 2).
To validate the clinical performance, we applied the newly established procedure to identify N. meningitidis from 51 CFS samples from N. meningitidis suspected patients. The standard conventional realtime PCR assay with MetA genes as a molecular target was also used to con rm the presence of N. meningitidis DNA. Realtime PCR identi ed N. meningitidis DNA from 13 out of 51 recruited CFS samples. Whereas, single-use of LAMP assay identi ed 18 cases positive with N. menitigitis, in which, ve cases did not match to either result acquired by real-time PCR or patients' clinical symptoms and were considered as false positive. However, when LAMP reaction mixtures from 51 CFS samples were treated with Crispr cas12a, only 13 cases were positive. Importantly, all of these 13 cases were matched to the results gained by conventional PCR (Fig. 2).

Discussion
Single-use of LAMP embeds high risk of false-positive signals that challenge the employing LAMP-based assays into clinical practices [7,9]. However, LAMP has strong intrinsic ampli cation potential and simple to operate, hence would bene t the communities. Our data revealed that the sequential treatment LAMP products by Crispr cas12a under the guidance of speci c gRNA sequence can abolish un-speci c signals. This technical integration of two enzymes, in one side help to sustain the strong ampli cation potential of LAMP on the other side, signi cantly enhances the speci city of diagnostic procedures.
We found that trehalose helps Crispr cas12a and Bst DNA Polymerase to sustain their activity in a single trehalose containing buffer at 55 o C. This condition omits buffer replacement from the LAMP into Crispr assay thereby reducing sample handling and contamination risk. However, in various tested biochemical environments, Crispr Cas12a strongly inhibits isothermal DNA polymerases (Bst and Bsu), therefore, we were not successful to couple isothermal enzymes and Crispr cas12 into single reaction tubes. Further studies are needed to mitigate the inhibitory effect of CRISPR Cas12a to Bst or Bsu DNA polymerase, thereby combining Crispr cas12a with isothermal ampli cation into single tube diagnostic device.
In conclusion: The sequential combination of LAMP ampli cation and Crispr cas12a treatment can alleviate false-positive acquired by single use of LAMP performance.

Declarations
Ethics statement: The study was submitted for regulatory approval to the Institutional Review Board of the 108 Military Central Hospital in Hanoi and was approved. The Ethical Committee of the 108 Military Central Hospital, Hanoi, provided ethical approval for the study. Informed written consent was obtained from all study participants or from their parents/guardians if the study participant was in an unconscious condition Availability of data and materials: Data and supporting materials associated with this study will be shared upon request

Competing interests
The authors declare no con ict of interests. Tables Table 1  Oligonucleotides