First application of loop‐mediated isothermal amplification (LAMP) assays for rapid identification of mating type in the heterothallic fungus Aspergillus fumigatus

Summary Background Loop‐mediated isothermal amplification (LAMP) assays, which operate at a single temperature and require no postreaction processing, have been described for rapid species‐specific detection of numerous fungi. The technology has much less commonly been applied to identification of other key genetic traits such as fungicide resistance, and has not yet been applied to mating‐type determination in any fungus. Objectives To develop first LAMP assays for mating‐type identification in a fungus, in this instance with the saprophytic mould and human opportunistic pathogen Aspergillus fumigatus, a heterothallic ascomycete requiring isolates of opposite mating type (MAT1‐1, MAT1‐2) for sexual reproduction. Methods New LAMP primer sets, targeted to MAT gene sequences, were screened against 34 A fumigatus isolates (of known mating type) from diverse clinical, environmental and geographic sources to establish whether they could distinguish MAT1‐1 or MAT1‐2 genotypes. Results and conclusions The new assays, operating at a single temperature of 65°C, correctly identified the mating type of A fumigatus isolates in <20 minutes, and thus have numerous research and practical applications. Similar MAT LAMP assays could now be developed for other fungi of agricultural, environmental, industrial and/or medical importance.


| INTRODUC TI ON
The fungus Aspergillus fumigatus is a saprophytic mould commonly found on plant debris and in soil. It is also an opportunistic human pathogen causing allergic symptoms and life-threatening invasive infections. The incidence of invasive aspergillosis (IA) has been increasing in recent years largely due to increased numbers of immunocompromised individuals in the population unable to fight off infection. 1 For more than 145 years, A fumigatus was only known to reproduce asexually, although several signatures of cryptic sexuality were present, for example, presence and expression of mating (MAT) genes and evidence of gene recombination within natural populations. 2 However, the breakthrough 2009 discovery of a functional sexual cycle 3 had several implications including: (a) potentially explaining high genotypic diversity observed in populations; (b) production of sexually derived airborne ascospores possibly more resilient to unfavourable environmental conditions; and (c) generation of sexual progeny with potentially greater pathogenicity and/or reduced sensitivity to fungicides. 4 Aspergillus fumigatus possesses a heterothallic (obligate outbreeding) mating system, with highly dissimilar stretches of DNA, termed "idiomorphs," present in isolates of opposite mating type as is characteristic for heterothallic ascomycete species. 5 Thus, MAT1-1 isolates contain an alpha-domain MAT1-1-1 gene whereas MAT1-2 isolates contain a high-mobility group MAT1-2-1 gene together with a recently described MAT1-2-4 gene. 6 A multiplex PCR-based assay for determination of mating type has previously been developed for A fumigatus. 2 More recently, loop-mediated isothermal amplification (LAMP) assays have become increasingly used for rapid species-specific detection of numerous fungi, including A fumigatus. 7 LAMP technology, first described by in 2000, 8 typically involves 4-6 primers in each reaction and has several purported advantages over PCR-based diagnostics. These include faster reaction times, potentially improved sensitivity and specificity, increased tolerance of sample inhibitors, no requirement for additional postreaction processing (eg, resolving PCR products on agarose gels) and use of only a single constant reaction temperature thus raising the possibility of field-based detection. Despite these advantages, LAMP assays have much less commonly been applied to detection of other key genetic traits such as fungicide resistance, one recent example being an assay targeted to a 34 bp tandem repeat in the cyp51A gene that has been associated with azole resistance in A fumigatus. 9 To date, however, LAMP assays have not been used for rapid detection of different mating types in fungi. The objective of the present study was therefore to develop and evaluate for the first time whether LAMP assays could be used for the rapid identification of mating type in a fungus, with a focus here on the human opportunistic pathogen A fumigatus.

| Ethics statement
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as the research in this article related to micro-organisms. DNA was quantified via nanodrop spectrophotometer and diluted to 10 ng/ μL using PCR-grade water. The mating type of these isolates was first determined using the published multiplex PCR assay (Table 1). 2 Amplicons were resolved on agarose gels, with 834 bp or 438 bp products amplified from MAT1-1 or MAT1-2 isolates, respectively.

| Development and validation of MAT LAMP assays
For all A fumigatus isolates tested, identical MAT genotype results were obtained using the previously described multiplex PCR assay 2 (see Figure 1 for representative results) and the new MAT-specific LAMP assays developed in the present study ( Figure 2, Table 2).
No-template (water) controls tested negative, that is, no amplification curves or dissociation plot peaks were observed (data not shown). and industrial [eg, Penicillium chrysogenum-used in penicillin pro-

CO N FLI C T O F I NTE R E S T
No conflict of interest is declared.

AUTH O R CO NTR I B UTI O N S
KMK, NJH, PSD, JSW and BAF conceived the ideas; KMK and SA collected the data; KMK analysed the data; KMK led the writing; all authors critically reviewed the manuscript prior to submission.