# Developing a genomic-based strategy to confirm microbial identity in bio-inputs containing multiple strains: an easy, fast, and low-cost multiplex PCR applied to inoculants carrying soybean Bradyrhizobium

## TL;DR

This paper introduces a fast and low-cost multiplex PCR method to identify multiple Bradyrhizobium strains in soybean inoculants, replacing a slower and more expensive technique.

## Contribution

A novel, genome-based multiplex PCR strategy for rapid and cost-effective identification of multiple Bradyrhizobium strains in microbial inoculants.

## Key findings

- The multiplex PCR method successfully identified four Bradyrhizobium strains in a single test.
- The new method reduced labor hours and costs by 3.08 and 3.25 times, respectively, compared to rep-PCR.
- Results were obtained in one day, versus 15 days for the traditional method.

## Abstract

Brazil stands out in research, industrial development, and farmers' use of microbial inoculants, with an emphasis on getting benefits from the biological nitrogen fixation process with the soybean crop. Nowadays, about 140 million doses of inoculants are commercialized annually for the soybean in the country, and strain identification is achieved by rep-PCR, an effective but time-consuming method. Aiming to develop an easy, low-cost, and low-time-consuming method, we used a complete genome-based approach based on the unequivocal identification of unique genes present in the genomes of each of the four Bradyrhizobium strains used in commercial inoculants: Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019, Bradyrhizobium japonicum SEMIA 5079, and Bradyrhizobium diazoefficiens SEMIA 5080. The unique pairs of primers able to amplify genomic regions of different sizes allowed the identification of the four strains in a simple multiplex polymerase chain reaction (PCR). Validation was confirmed by using single colonies, multiple cultures, and commercial inoculants. The number of labor hours of a technician was 3.08 times higher, and the final cost was 3.25 times higher in the rep-PCR than in the multiplex PCR. Most importantly, the results for multiplex PCR were obtained on the same day, in contrast with 15 days in the traditional methodology. The genomic approach developed can be easily applied to a variety of microbial inoculants worldwide, in addition to studies of ecology and evaluation of the competitiveness of the strains.

The online version contains supplementary material available at 10.1007/s42770-024-01441-8.