Development of a dry-reagent-based qPCR to facilitate the diagnosis of Mycobacterium ulcerans infection in endemic countries

Abstract

Background: Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR) is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR) diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection.

Methodology/principal findings: We compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i) highly resistant to heat; (ii) of similar sensitivity and efficiency to the fresh mix and (iii) easier to use than the fresh mix.

Conclusions: Dry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field.

Fig 1. Diagram of the study.

Fig 2. Comparison between 3 dry-reagent conditions and the standard reference method, based on an external standard curve.

Serial 10-fold dilution from 1x10⁶ U/ml to 1x10⁰U/ml of M. ulcerans DNA. The serial curve was generated by a linear regression of the threshold cycle (C_t) against the logarithm of M. ulcerans DNA concentration. Each 10-fold dilution was performed in triplicate. One standard deviation to either side of the mean is shown. Standard QPCR Mix refers to the reference method with fresh mix (y = −3.4107x + 40.357); dry-mix condition A refers to the dry mix containing 5% cryo T and 1% cryo P (y = −3.2333x + 39.087); dry-mix condition B refers to the dry mix containing 10% cryo T and 1% cryo P (y = −3.3727x + 39.942); dry-mix condition C refers to the dry mix containing 7.5% cryo T and 0.5% cryo P (y = −3.357x + 39.851). For each assay, the coefficient of correlation was greater than 0.99.

Fig 3. Comparison of the 3dry-reagents conditions after exposure to a temperature of 50°C for 15 days and the standard reference method, based on an external standard curve.

Serial 10-fold dilutions, from 1x10⁶ U/ml to 1x10² U/ml, of M. ulcerans DNA. The serial curve was generated by linear regression of the threshold cycle (C_t) against the logarithm of M. ulcerans DNA concentration. Each 10-fold dilution was performed in triplicate. One standard deviation on either side of the mean is shown. “Standard QPCR Mix” corresponds to the reference method with fresh mix (y = −3.34x + 39.88); dry mix A is the dry mix containing 5% cryo T and 1% cryo P (y = −3.23x + 43.94); dry mix B is the dry mix containing 10% cryo T and 1% cryo P (y = −3.26x + 38.98); dry mix C is the dry mix containing 7.5% cryo T and 0.5% cryo P (y = −3.19x + 38.55).

Fig 4. CT values for the 19 weakly positive samples, for 2 conditions.

(A) C_t results for each sample obtained by the fresh mix and the dry mix B, Horizontal bars indicate mean values ± SD. (B) Mean values ± SD and average ΔC_t ± SD between fresh mix and dry mix B.

Fig 5. CT values for the 55 positive samples, for 3 mix conditions.

(A) C_t results for each sample obtained by the fresh mix and the dry mix B in vial or in strip format, Horizontal bars indicate mean values ± SD. (B) Mean values ± SD and average ΔC_t ± SD between fresh mix and dry mix B in vial or in strip format.